Acetylcholinesterase (ache)-derived peptide as an inducer of granulocytopoiesis, uses and methods thereof

ABSTRACT

The present invention describes the use of an AChE-R-derived peptide, also known as ARP, as an inducer of hemopoietic cell differentiation and expansion, specifically for the granulocytic population. In addition, the use of ARP as an inducer of thrombopoietin and pro-inflammatory cytokines is also presented. ARP may further be used in the pre-transplant priming of hematopoietic stem cells. Other uses and methods utilizing ARP are also described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The instant application is a divisional of application Ser. No.10/589,116, filed on May 1, 2007, which is the U.S. national stageapplication of International Application No. PCT/IL2005/000185, filed onFeb. 10, 2005, the entire disclosures of which are herein incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of hematopoiesisand more specifically to the effect of an AChE-derived peptide ondifferent hematopoietic sub-populations.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Mammalian hematopoietic stem cells develop during embryogenesis anddifferentiate into the different hematopoietic lineages. After birth,the capacity of myeloid cells to respond to external and/or internalstimuli by the finely tuned production of pro-inflammatory andanti-inflammatory cytokines is gradually acquired, in parallel with theestablishment of fully mature lymphocytic immune responses.Interestingly, the responses of both myeloid and lymphoid cell lineagesare subject to acetylcholine (ACh) modulation [Kawashima, K., and T.Fujii (2000) Pharmacol. Ther. 86:29-48; Tracey, K. J. (2002) Nature420:853-9], which involves the α7 nicotinic ACh receptor [Wang, H. etal. (2003) Nature 421:384-8] and are known to be impaired underpsychological stress [Miller, G. E. et al. (2002) Health Psychol21:531-41]. However, the putative protein(s) mediating thesedevelopmental and stress-induced processes is yet unknown.

Post-stress leukocytosis, i.e. overproduction of white blood cells(WBC), was first described over 50 years ago. Elevated WBC counts occurafter diverse stress insults, e.g. shock, blood loss, in post-partummothers, following space flight or bacterial infection [Delgado, I. etal. (1994) Gynecol. Obstet. Invest. 38: 227-235; Reizenstein, P. (1979)Br. J. Haematol. 43: 329-334; Stowe, R. P. et al. (1999) J. Leukoc.Biol. 65: 179-186; Toft, P. et al. (1994) Apmis 102: 43-48; Wanahita, A.et al. (2002) Clin. Infect. Dis. 34: 1585-1592]. The initiation of WBCoverproduction has been attributed to the elevated serum levels ofcortisol, causing both enhanced proliferation and facilitated WBCmaturation, predominantly toward the granulocytic lineage [Abramson, N.and Melton, B. (2000) Am. Fam. Physician 62: 2053-2060]. However, theincreased levels of cortisol, e.g. following the stressful event ofdelivery, recede within a few hours [Tuimala, R. et al. (1976) Br. J.Obstet. Gynaecol. 83: 707-710], and cannot account for the prolongationof leukocytosis, especially since the lifespan of granulocytes isextremely short, with 50% of the granulocytes being replaced by the bonemarrow daily [Abo, T. and Kawamura, T. (2002) Ther. Apher. 6: 348-357].The signaling pathways controlling this process therefore remain largelyunknown.

Granulocytosis depends upon the production ofpro-inflammatory/hematopoietic cytokines which in peripheral tissues isregulated by acetylcholine (ACh) [Borovikova, L. V. et al. (2000) Nature405: 458-462; Tracey, K. J. (2002) id ibid.]. Under normal conditions,ACh activates α7 ACh nicotinic receptors on macrophages to attenuatepro-inflammatory cytokine secretion at the post-transcriptional level[Wang, H. (2003) id ibid.]. To determine whether post-stress ACh levelscan account for the prolonged granulocytosis effect independently ofcortisol, and to delineate the cascade of events that enables thisprocess, the inventors studied circulating acetylcholinesterase (AChE).Agents performing this reaction can further be used to control theproduction of cytokines in patients with failure of such responses.

Hence, inflammation is an example of inducible hematopoiesis, whichoccurs whenever there is an increased demand for mature blood cells.Upon activation of the inflammatory response, pro-inflammatory cytokinesare secreted by cells of the immune system, and induce acceleratedproduction of hematopoietic cells. Lipopolysaccharide (LPS), the maincell wall component of gram-negative bacteria, is an endotoxin thatinduces an acute inflammatory response, initiating a signal transductioncascade that leads to the release of inflammatory cytokines, whichinclude tumor necrosis factor (TNF)-α, IL-1β, IL-6 and IL-8. Thesecytokines activate the mobilization of hematopoietic cells from the bonemarrow (BM) and set in motion the migration of leukocytes from bloodvessel walls, increasing their numbers in the circulation [Lagasse E,Weissman I L. (1996) J. Immunol. Methods 197:139-150]. The net result ofthis process is an immediate and dramatic increase in the number ofcirculating peripheral blood (PB) cells, needed to mount the immuneresponse. This results in a compensatory decrease in cell numbers untilmore cells are produced in the BM [Nagata Y, et al. (1997) ThrombHaemost. 77:808-814].

Many factors are involved in abating the inflammatory response allowinghemostasis to return. Acetylcholine (ACh), is one of the recentlydiscovered factors that attenuates the pro-inflammatory cytokinesecretion by activating nicotinic receptors on macrophages at thepost-transcriptional level [Wang H. et al. (2003) Nature 421:384-388].Circulating acetylcholinesterase (AChE) controls the levels of ACh,suggesting promotion of the inflammatory process under AChE excess [PickM. et al. (2004) Ann. NY Acad. Sci. 1018:85-95]. AChE has three variantforms, —Synaptic (S), Erythrocytic (E) and Readthrough (R), isubiquitously expressed in hematopoietic cell lineages especially inmegakaryocytes (Mks) and erythrocytes [Kawashima K, and Fujii T. (2000)Pharmacol. Ther. 86:29-48; Lev-Lehman E. et al. (1997) Blood89:3644-3653; Grisaru D. et al. (2001) Molecular Medicine 7:93-105] andis thought to be a potential growth factor for hematopoiesis [Grisaru(2001) id ibid.; Deutsch V. et al. (2002) Exp. Hematol. 30:1153-1161].

AChE-R is expressed in multiple embryonic and tumor cells, where itdisplays morphogenic functions, but it is rarely found in healthy andunstressed adult tissues [Grisaru (1999a) id ibid.; Karpel (1994) idibid.; Soreq, H. and S. Seidman (2001) Nat. Rev. Neurosci. 2:294-302;Grisaru, D. et al. (1999b) Mol. Cell Biol. 19:788-795] or human sera[Brenner et al. (2003) FASEB J. 17(2):214-22]. Cortisol induces AChE-Rproduction in cultured CD34+ blood cell progenitors [Grisaru (2001) idibid.], while ARP₂₆, a synthetic peptide designed to mimic the cleavableC-terminal sequence of AChE-R, promotes hematopoietic proliferation invitro [Grisaru (2001) id ibid.].

The up regulation of AChE expression during megakaryopoiesis wasinitially reported in rats, where the fraction of AChE-positive BM cellsincreased following induction of thrombocytopenia [Jackson C W. (1973)Blood 42:413-421]. Functional involvement of this enzyme was indicatedby suppression of AChE synthesis, which induced transient decreases inmurine megakaryocyte progenitors [Lev-Lehman (1997) id ibid.].

Platelet production is a self-regulated process primarily induced bythrombocytopenia where a drastic reduction in platelets stimulates theproduction of thrombopoietin (TPO). Subsequently, as platelet countsreturn to normal, TPO is effectively cleared from the circulation, bymeans of binding to its receptor, c-mpl, and uptake into platelets andmegakaryocytes. TPO is the main physiological growth factor formegakaryocyte proliferation, differentiation and platelet production.Nevertheless, c-mpl^(−/−) and TPO^(−/−)knockout mice have a residual 10%of normally functioning megakaryocytes and platelets, which cannot beattributed to IL-6, IL-11 or leukemia inhibitory factor (LIF), which arealso known to induce megakaryocyte differentiation [Ishibashi T. et al.(1989) Proc. Natl. Acad. Sci. USA 86: 5953-5957; Teramura M., et al.(1996) Cancer Chemother. Pharmacol. 38:Suppl:S99-102; Nakashima K. etal. (1998) Semin. Hematol. 35: 210-221; Gainsford T. et al. (2000) Blood95: 528-534] suggesting the involvement of other factor(s) in thisprocess.

Pancytopenia and prolonged thrombocytopenia are significant clinicalproblems for patients undergoing BM transplantation. Engraftment oftransplanted BM is usually accomplished within 2 to 3 weeks, duringwhich period the patient is susceptible to life-threatening infectionsand bleeding. Platelet recovery after autologous stem cells or cordblood (CB) transplantation is significantly delayed (up to 6 weeks posttransplant) due to lack of sufficient megakaryocyte precursors in thegrafts. The paucity of megakaryocyte progenitor cells in grafts, and notinferior levels of TPO, is the cause for delayed platelet recoveryobserved post cord blood and autologous transplantation [Kuter D. J.(2002) Transfusion 42:279-283; Kanamaru S. et al. (2000) Stem Cells18:190-195].

Thus, within their individual microenvironment, blood cells receive aplethora of external stimuli which influence transcription andprocessing of many reactive molecules. Particular alternatively splicedAChE variants may be candidates to exert both enzymatic andnon-catalytic functions on these cells. The expression of AChE-S inblood cells has been associated with terminal differentiation [Chan, R.Y. Y. et al. (1998) J. Biol. Chem. 273:9727-9733] and apoptosis [Zhang,X. J. et al. (2002) Cell Death Differ. 9:790-800]. In contrast, AChE-Rand the synthetic peptide ARP were associated with stem myeloid cellproliferation [Grisaru (2001) id ibid.; Deutsch et al. (2002) id ibid.].

The present inventors performed a comprehensive study to correctlyevaluate the potential contribution of AChE towards differentiation,proliferative or apoptotic events in hematopoiesis, and in inflammatoryresponses under stress stimuli, specific variants were identified, theirlevels quantified and their subcellular localization (i.e. on the cellsurface and/or intracellular) determined in specific blood celllineages.

The inventors considered, as a working hypothesis, circulating AChE-R tobe a modulator of sustained granulocytosis effects in hematopoieticprogenitors. To find out whether AChE-R and/or ARP are associated withpost-stress granulocytosis and cytokine production, the inventorsinitiated a study aimed at delineating the in vivo and ex vivoregulation of AChE-R production in stress-induced myelopoieticprocesses.

Thus, an aim of the present invention is to provide novel uses for anAChE-derived peptide, as an agent capable of inducing granulopoiesis, asdemonstrated in the following Examples.

It is another aim of the present invention to provide a method for thetreatment of conditions that induce a low granulocytic cell count,administering said AChE-derived peptide, and compositions thereof, to asubject in need.

Further, the present invention provides methods of evaluatinglymphocytic activity, based on the expression of the different AChEforms on lymphocytes.

Other purposes and advantages of the invention will appear as thedescription proceeds.

SUMMARY OF THE INVENTION

The inventors have demonstrated that overproduction and C-terminalcleavage of the stress-induced AChE-R isoform induced granulocytosis.

In this view, in a first aspect, the present invention provides the useof an AChE-derived peptide, ARP₂₆, and any functional fragments thereof,as an agent for the induction of the production of granulocytes, or forthe enrichment of the granulocytic cell population, wherein said peptideis denoted by SEQ ID NO:1. The peptide used by the invention comprisesthe following amino acid sequence:

N′-GMQGPAGSGWEEGSGSPPGVTPLFSP-C′

Said peptide may also be an agent for the induction of repopulationand/or rematuration of granulocytic cell populations, preferably in asubject in need.

In another aspect, the present invention comprises the use of anAChE-derived peptide as an agent for ex vivo or in vitro manipulation ofcells to induce granulocyte cell differentiation, wherein said peptideis denoted by SEQ ID NO:1.

The AChE-derived peptide denoted by SEQ ID NO:1, or any functionalfragments thereof, are also to be used as an agent for pre-transplantpriming of hematopoietic stem cells.

A further use of the AChE-derived peptide ARP₂₆, or any functionalfragments thereof, is as an inducer of pro-inflammatory cytokines and/oras an inducer of TPO.

In a further aspect, the present invention provides the use of anAChE-derived peptide, or any functional fragments thereof, in thepreparation of a pharmaceutical composition for the treatment and/orprevention of conditions that trigger low granulocyte count, whereinsaid peptide is denoted by SEQ ID NO:1. Said composition may also beused in pre-transplant priming of hematopoietic stem cells. Saidconditions may be, for example, leucopenia, acute myeloid leukemia(AML), and particularly neutropenia.

In an even further aspect, the present invention provides a method oftreatment of conditions that induce leucopenia, comprising the steps ofadministering a therapeutically effective amount of an AChE-derivedpeptide or a composition thereof to a subject in need, wherein saidAChE-derived peptide is denoted by SEQ ID NO:1.

The invention also refers to an in vivo method for the prevention and/ortreatment of conditions wherein lymphocyte activity is reduced, such aschronic stress, autoimmune diseases, inflammation, rheumatoid arthritis,multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),fibromyalgia, multiple chemical sensitivity, post-irradiation,chemotherapy in a subject in need, comprising administering atherapeutically-effective amount of an AChE-derived peptide, or anyfunctional fragments thereof, to an individual suffering or prone tosaid conditions, wherein said peptide is denoted by SEQ ID NO:1.

The present invention also discloses a method for detecting changes inthe activity of lymphocytes, comprising measuring the expression ofAChE-R on the surface of lymphocytes.

The invention provides an ex vivo or in vitro method of preventionand/or treatment of conditions wherein lymphocyte activity is reduced,such as chronic stress, autoimmune diseases, inflammation, rheumatoidarthritis, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),fibromyalgia, multiple chemical sensitivity, post-irradiation,chemotherapy in a subject in need, comprising obtaining blood from saidsubject, isolating immature cells and contacting said cells with anAChE-derived peptide, or any functional fragments thereof, wherein saidpeptide is denoted by SEQ ID NO:1.

In addition, a method of priming of hematopoietic stem cellspre-transplant is presented, comprising obtaining said cells, isolatingfrom said cells an immature, CD34+ rich population, and exposing saidcell population to an AChE-derived peptide, its functional fragments orderivates, or compositions comprising thereof, wherein said peptide isdenoted by SEQ ID NO:1. Most importantly, said cells may be obtainedfrom the subject in need of said transplant or from another donor.

Lastly, the invention also provides a method of inducing adult bloodcells to produce cytokines, comprising obtaining said cells from asubject in need of cytokine-producing blood cells, isolating immaturecells and contacting said cells with an AChE-derived peptide, whereinsaid peptide is denoted by SEQ ID NO:1. This method is particularlyadvantageous for patients with neutropenia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D: Flow cytometric approach to AChE splice variants.

FIG. 1A: C-terminal amino acid sequence unique to the human AChE-Svariant; SEQ ID NO:2.

FIG. 1B: C-terminal amino acid sequence unique to the human AChE-Rvariant; SEQ ID NO:1. The sequences in A and B share a similar coredomain. Note that ASP, but not ARP, includes a C-terminal cysteineresidue (asterisk) that enables AChE-S multimerization).

A scFv-myc tagged antibody selected against the C-terminal sequence ofAChE-S from a phage display library (anti-ASP1) and a polyclonalantibody produced against synthetic ARP (drawings) enabled specificdetection of each of these variants.

FIG. 1C: Flow cytometric sub-classification of hematopoietic cells usinganti-CD45. Shown are adult peripheral blood cells divided intolymphocytes, monocytes, granulocytes and red blood cells, depending ontheir expression of CD45. Each dot corresponds to one cell.

FIG. 1D: Anti-ASP1 scFv purity was verified by gel electrophoresis.Elution from Ni-NTA column with 250 mM imidazole, revealed 30-kDa band(arrow). Abbreviations: S. Sc., side scatter; fr., fraction.

FIG. 2A-2C: Enzymatic AChE activity in hematopoietic blood cells.

FIG. 2A: Cytochemical staining reveals acetylthiocholine hydrolysisactivity (brown-gray) in all cell lineages (arrows) from all threesources.

FIG. 2B: Counterstaining with May-Gruenwald's/Giemsa highlights thedifferent characteristic morphologies of the smeared cells. Note graycolor of cytochemically positive cells (arrows).

FIG. 2C: Quantitation of cell positive for AChE activity for each bloodcell group and arbitrary measurement of brown intensity n=30 cells.

Abbreviations: L, lymphocytes; G, granulocytes; R, red blood cells; M,monocytes; pos., positive; int., intensity.

FIG. 3: Cell surface and intracellular AChE-S and -R labeling inpost-partum peripheral blood cell populations.

AChE-S and AChE-R were detected using an anti-ASP scFv antibody with amyc tag and anti-myc FITC or a polyclonal rabbit antibody andanti-rabbit FITC, respectively. Positive cells (solid line) were definedby a shift to the right as compared to the control (dashed line)histogram. This figure represents one of 15 reproducible analyses.

Abbreviations: C, Cytoplasmic; S, Surface; rbc, red blood cells; gran.,granulocytes; mono., monocytes; lymph., lymphocytes; ce. no., cellnumber; Fluor. Int., Fluorescence Intensity.

FIG. 4A-4C: Immunochemical analyses of cord blood cell lysates. AChE-Sand -R epitopes were detected in 0.83 mg/ml protein extracts from thethree different sub-populations of cord blood.

FIG. 4A: Plasmon resonance traces reflected interactions of anti-ASP1scFv antibody with extracts of granulocytes (G); lymphocytes (L);monocytes (M) and red blood cells (R). Real time interaction (x-axis) isexpressed in RU (y-axis).

FIG. 4B: Shown are the relative contents of AChE-S and -R expressed asRU between the respective antibodies and their epitopes in the differentcell lysates, standardized to the amount of total protein in the extract(top) or the number of lysed cells (bottom).

FIG. 4C: Immunoblot using the phage anti-ASP1 and lysates of the notedcell populations. AChE-S and its various cleaved products are labeled.PC12 cells known to express AChE-S were used as a positive control.

Abbreviations: T., time; prot., protein; ce., cells.

FIG. 5A-5B: Development- and stress-associated expression of AChEvariants within blood cell populations.

Positive cell fractions were quantified by flow cytometry and dividedinto cells with the corresponding variants in the cytosol and on thesurface of the noted cell populations. Columns present the percent ofpositive cells in 15 samples from each source (mean±standard error ofthe mean). Nonspecific signals were subtracted. Solid and crosshatchedbars represent cytoplasmic and surface expressions, respectively.

FIG. 5A: AChE-S

FIG. 5B: AChE-R

Abbreviations: R, red blood cells; G, granulocytes; M, monocytes; L,lymphocytes; Pos., positive; ce., cells; tot., total; c., cytoplasmic;s., surface; C.B., cord blood; Ad. B., adult blood; PPB; post-partumblood.

FIG. 6A-6B: Surface AChE-R on lymphocyte subpopulations.

FIG. 6A: Surface AChE-R was detected in T and B lymphocytesub-populations on all three sources analyzed. Shown are cells fromadult peripheral blood. Upper panel is surface AChE-R expression on Tcells and lower expression on CD19+CD45+ B lymphocyte. Backgroundstaining (dashed curve) and surface AChE-R staining (bold curve).Insert: B cells were defined by their high expression of the pan-Bmarker CD19 (y-axis) and CD45 (x-axis). T cells were labeled with thepan-T marker CD3 together with CD45.

FIG. 6B: Surface AChE-R contents on lymphocyte sub-populations. Anaverage of ten samples from each source of cells were used. Shown aremean percent values of cells expressing surface AChE-R, and the meanfluorescent intensity (MFI)±standard error of the mean. Significantdifferences values (t-test, p<0.05) are marked by an asterisk.

Abbreviations: C, cord blood; A, adult peripheral blood; P,post-delivery peripheral blood.

Abbreviations: Ce. co., cell count; fluor. U.; fluorescence units; pos.,positive.

FIG. 7A-7E: Spatiotemporal shifts in embryonic AChE mRNAs within bloodcell forming tissues.

FIG. 7A: Schematic of the human ACHE gene and its alternative mRNAs. Thecore of human AChE is encoded by three exons, and parts of additionalregions encode the variant-specific C-terminal sequences. Transcriptionbegins at E1, and E2 encodes a leader sequence that does not appear inany mature protein. In addition to a proximal promoter (red lineadjacent to E1), a distal enhancer region (the other red line) is richin potential regulatory sequences, some of which are shown as wedges.

FIG. 7B: Sagital section of a human embryo showing the hematopoieticorgans—AGM (aorta-gonad-mesonephros, blue), LIV (liver; green), SPL(spleen; red), and BM (bone marrow; brown).

FIG. 7C: Scheme of gestational shifts in hematopoietic processes showsthe relative levels of blood cell formation in the various hematopoieticorgans throughout human gestation [Tavassoli, M. (1991) Blood Cells17:269-281]. Ages for which in situ hybridization was performed aremarked by gray columns.

FIG. 7D: Representative in situ hybridization in liver micrographs fromhuman fetuses at the noted gestational ages. Selective probes for eachof the alternative human AChE mRNA transcripts showed increasedexpression (red precipitate) of AChE-R mRNA at 16 weeks of gestation, atthe same time when the liver changes from erythropoiesis tomyelopoiesis.

FIG. 7E: Line colors representing (as in 7C) spatiotemporal changes inlabeling intensity and standard error of the mean (SEM) for each probeand organ, expressed as percentage of red pixels in each slide [Grisaru(1999a) id ibid.]. Note that AChE mRNA expression increases parallel toactive hematopoiesis in the examined organs (N=4-6 tests for each organin each gestational age). mRNA peaks in the liver at 16 weeks,coinciding with a shift in fetal liver hematopoiesis, fromerythropoiesis to myelopoiesis [Porcellini, A. et al. (1983) Int. J.Cell Cloning, 1: 92-104].

Abbreviations: G. a., gestational age; wk., weeks; r.p., red pixels.

FIG. 8A-8G: Parturition-induced transient increases in cortisol andsustained increases in catalytically active plasma AChE- andAChE-R-positive granulocytes.

FIG. 8A: Serum cortisol levels were higher than normal in the pre- andintra-partum periods. Note the significant decrease post-partum.

FIG. 8B: Plasma catalytic activity of AChE shows stable increase duringthe entire peri-partum period.

FIG. 8C: Alternative splicing of the ACHE gene.

FIG. 8D: Cortisol levels in patients during parturition (N=20) weresignificantly higher than in controls (Cont N=48), reflecting the stressof parturition.

FIG. 8E: Cortisol levels show direct correlation to increases in whiteblood cells (WBC) during parturition.

FIG. 8F: Increase in serum cortisol.

FIG. 8G: Intra-partum AChE-R-positive granulocytes (Gran) increases as afunction of the increase in serum cortisol.

Asterisks indicate statistical significance.

Abbreviations: cort., cortisol; act., activity; ce., cells.

FIG. 9A-9D: Peri-partum blood profile.

Shown are blood profile changes in patients before (PRE), during (INTRA)and following (POST) delivery. Dotted areas represent normal blood countranges.

FIG. 9A: WBC counts increase during labor (above normal range) anddecrease post-partum, albeit remaining above normal. Hemoglobin levels(Hgb) decrease below normal range during and after delivery. Platelet(Plat) counts remain stable (at normal range) during the entire period.

FIG. 9B: Sustained leukocytosis correlates with elevation in granulocyte(Gran), but not monocyte (Mono) or lymphocyte (Lymph) counts whichremained within normal range. Asterisks indicate statisticallysignificant differences (N=16 patients).

FIG. 9C: Shown are CD15 and CD33 labeling on AChE-R positivegranulocytes (upper panel) and CD14 and CD33 in monocytes (lower panel).Note decreases in CD15 expression in intra-partum granulocytes anddecreases in post-partum monocytes.

FIG. 9D: The significant intra- and post-partum increase in AChE-Rpositive granulocytes, but not monocytes or lymphocytes, may explain thestable serum activity. Immunoblots (insert) of serum proteins from 3patients demonstrate AChE-R presence. Luminiscence analysis of theAChE-R blot (upper insert) shows stable presence of AChE-R in the serumof women during the peri-partum period.

Abbreviations: Tot., total; MFI, Mean Fluorescence Intensity; Gran.,granulocytes; momo., monocytes; exp., expression; ce., cells.

FIG. 10: Stress induced AChE-R in white blood cells correlates withpresence of active AChE-R in the plasma.

The figure shows a direct correlation between AChE-R expression in alltypes of white blood cells (granulocytes, monocytes and lymphocytes) andits activity in the plasma of post-partum mothers.

FIG. 11A-11E: ARP₂₆ operates as an inducer of ACHE gene expression andpotentiates myeloid expansion in vivo.

FIG. 11A: Structure of the AChE-R isoform with the stress inducedcleavage (arrow) of the C-terminus (ARP).

FIG. 11B: Human cord blood CD34⁺ cells treated for 24 hours with thenoted doses of ARP₂₆ as the sole growth factor were subjected to in situhybridization with probes selective for each of the noted AChE mRNAsplice variants. Shown are representative micrographs of the cells.Lower panels: Cytochemical staining for AChE catalytic activity in thepresence of 10⁻⁵ M iso-OMPA, a selective inhibitor ofbutyrylcholinesterase (center) and nuclear staining with DAPI (bottom).Note intensified brown precipitates of AChE reaction product, mainlyunder 2 nM of ARP₂₆.

FIG. 11C: Average labeling densities for 10-20 individual cells. Notethe concomitant increases in all transcripts, peaking at 2 nM ARP, andthe limited variance between cells.

FIG. 11D-11E: Flow cytometric analysis of CD34+-derived hematopoieticcells after 2 weeks in liquid culture. Incubation with ARP₂₆, but notwith cortisol, ASP₄₀ or PBAN, increased the total number of cells. FIG.11D: The expansion index (the number of viable cells/ml culture dividedby the number of seeded cells) was considerably higher followingincubation with ARP₂₆. FIG. 11E: The percentage of immature stem cells(left column), committed myeloid cells (middle column) and maturemyeloid cells (right column) that developed in the presence of eachsupplement is indicated by numbers on the relevant dot plots. Unlabeledcells appear as black dots and double-labeled ones as green dots. Notesimilar patterns under the influence of cortisol and ARP₂₆, but not ofthe ASP₄₀ and PBAN negative control peptides.

Abbreviations: pix., pixels; c.e.i., cell expansion index; treat.,treatment; I.S.C., immature stem cells; com. My., committed myeloid;mat. my., mature myeloid; fluoresce., fluorescence; u., units; cont.,control; iso., isotype; cort., cortisol.

FIG. 12A-12C: ARP induces cytokine elevation in WBC.

FIG. 12A: Plasma cytokine levels in intra-partum patients and matchedcontrols, measured by a particle-based flow cytometry immunoassay (humaninflammation cytometric bead array kit, BD Bioscience, Palo Alto,Calif.). Note elevation of IL-12, IL-6, and IL-1β under post-partumconditions (N=15 in each group).

FIG. 12B: The proposed concept involves stress-induced elevation ofplasma cortisol, which promotes AChE-R overproduction in peripheralmononuclear cells. C-terminal cleavage of AChE-R yields ARP, whichamplifies AChE-R overproduction independently of cortisol. Accumulationof AChE-R potentiates ACh hydrolysis, alleviating the nicotinic α7 AChEcontrol over pro-inflammatory cytokine production and resulting inelevated TNFα and IL-6 (fluorescence intensity). Inset: Fluorescenceprofiles of IL-6 and TNFα-positive cells from ARP-treated (top) andcontrol culture (bottom).

FIG. 12C: To test causal relationship between elevated AChE-R andcytokine plasma levels adult peripheral mononuclear cells (N=3) wereincubated for 24 hours with or without 2 nM ARP₂₆. Note significantincreases in IL-6, IL-10 and TNFα levels, but not the anti-inflammatorycytokine IL-12, following ARP₂₆ treatment. Asterisks denotestatistically significant differences compared to control.

Abbreviations: Fluor., fluorescence.

FIG. 13A-13B: Expression pattern of transcription factors pivotal forhematopoiesis following inflammatory stress.

FIG. 13A: Relevant hematopoiesis related transcription factors bindingsites on the ACHE promoter.

FIG. 13B: Shown are expression of transcription factors pivotal forhematopoiesis in bone marrow extracts from FVB/N (dashed line) and TgRmice (solid line) (n=25), at different time points post LPS injection.Asterisks denote significant differences and results are presented asmean+SD (p<0.02, n=10), by real time RT-PCR. levels of transcriptionfactors levels in While the response pattern to LPS of LMO2, GATA1,RUNX1 and STAT5 was similar in both FVB/N and TgR mice, PU.1 levelsdecreased significantly in FVB/N but not in TGR mice bone marrow, inresponse to LPS. At 72 h post LPS injection, PU.1 levels recovered andeven reached higher than base-line values in FVB/N mice, but showed somedecrease in TgR mice.

Abbreviations: No., none; Fo. Dif., fold difference.

FIG. 14A-14B: Rapid post-LPS hematopoietic recovery in TgR mice.

FIG. 14A: Immunophenotyping of the hematopoietic progenitors and therelevant transcription factors during the differentiation.

FIG. 14B: Shown are WBC counts in FVB/N (dashed line) and TgR mice(solid line) (n=25). Results of morphological examination of TgR andFVB/N mice peripheral blood smears, at different time points post LPSinjection. Asterisks denote significant differences and results arepresented as mean+SD (p<0.02, n=10).

Peripheral blood immunophenotyping revealed that while FVB/N mice had asignificant decrease in GR1+ (granulocyte) cells, in response to LPSinjection, the number of GR1+ remained unchanged in TgR mice and wassignificantly higher than FVB/N by 72 h post LPS injection. Both FVB/Nand TgR mice had decreased CD11b+ (monocytic) cell counts 24 h post LPSinjection, although the decrease was steeper in TgR as compared to FVB/Nmice. CD11b+ cell counts recovered almost completely by 72 h post LPSinjection in both FVB/N and TgR mice, TgR mice attaining higher Cd11b+counts, although not reaching a statistically significant value.

Asterisks denote significant differences and results are presented asmean+SD.

Abbreviations: pos. ce., positive cells; no., none.

FIG. 15A-15C: Transgene facilitation of hematopoietic regulators.

FIG. 15A: Schematic of the proposed mechanism, which shows how astress-induced switch from production of AChE-S to the -R variantresults in hematopoietic progenitor cell expansion towards themegakaryocyte lineage and increased platelet counts.

FIG. 15B: Human (h) AChE-R DNA construct inserted into the FVB/N mousegenome for generating the TgR transgenic mice. hAChE-R cDNA-derived 100base pair product was successfully amplified in bone marrow DNA of TgR(5^(th) and 6^(th) lanes, after the marker—M), but not TgS or FVB/N mice(n=12, left arrow). A mouse actin product (130 base pair, right arrow)appeared in all 3 tested lines, FVB/N, TgR and TgS.

FIG. 15C: Levels of the pro-inflammatory cytokine IL-6 (pg/ml) and theAChE catalytic activity (activity per minute per gram of protein) in PBof TgR and FVB/N mice. Asterisks denote significant differences andresults are presented as mean±SD (p<0.01, n=10).

Abbreviations: h., human; m. act., mouse actin; PB, peripheral blood.

FIG. 16A-16D: Shorter post-LPS hematopoietic recovery in TgR mice.

Graphs show RBC×10⁹ (FIG. 16A), WBC×10⁶ (FIG. 16B) and platelet(Plts×10⁶) (FIG. 16C) counts per ml of FVB/N (dashed line) and TgR mice(solid line) (n=25) peripheral blood.

FIG. 16D: Results of morphological examination of TgR and FVB/N miceperipheral blood smears, at different time points post LPS injection asindicated. Asterisks denote significant differences and results arepresented as mean±SD of WBC×10⁶ per ml of blood (p<0.02, n=10).

Abbreviations: T. po., time post; gran., granulocytes; mono., monocytes;lymph., lymphocytes.

FIG. 17A-17B: Changes in TPO levels in response to LPS injection.

Thrombopoietin (TPO) levels were measured in bone marrow (BM) celllysates (FIG. 17A) and plasma (FIG. 17B) from TgR and FVB/N mice.Asterisks denote significantly different values. Results are presentedas mean±SD (p<0.04, n=10).

Abbreviations: po., post; ce. ly., cell lysates; plas., plasma.

FIG. 18A-18C: Facilitated progenitor cells potential in TgR mice.Committed colony-forming units of megakaryocyte (CFU-Mk, FIG. 18A),granulocytic/monocytic (CFU-GM, FIG. 18B) and multi-potential (CFU-GEMM,FIG. 18C) progenitors were quantified in a semisolid colony formationassay. Asterisks denote significantly different values. Assays were setup in triplicates from bone marrow preparations from 4 separate mice pertime point. Values represent mean±SD.

Abbreviations: T. po., time post.

FIG. 19A-19F: AChE-R, RACK1 and PKCε expression in megakaryocytes.

Bone marrow smears were stained with May-Grünwald (FIG. 19A) andspecific antibodies to detect AChE-R (FIG. 19B), RACK1 (FIG. 19C) andPKCε (FIG. 19D). The α symbol represents “anti”, meaning the antibodyagainst that specific protein was used in the respective staining.

FIG. 19E: Illustration of the putative interaction between the threeproteins.

FIG. 19F: Population distributions of Mk labeling intensities forAChE-R, RACK1 and PKCε. White bars represent FVB/N and grey, TgR mice BMlabeling intensities. Note the shift to the right, indicating increasedlevels of these 3 proteins in Mks from TgR as compared with FVB/N mice.n=50 cells per labeling experiment.

Abbreviations: Hist., histochemistry; ce., cells.

FIG. 20A-20B: Enhanced human cell engraftment with ARP₂₆. 100,000 humanCB CD34⁺ cells were injected into the tail vein of pre-irradiatedNOD/SCID mice with no priming of cells (none, white diamond symbol), orfollowing priming of cells with ARP₂₆ for 2-4 hours and injection withhuman ARP₂₆ (black square symbol) or ASP₄₀ (gray triangle symbol). Bonemarrow was harvested 6 weeks post-transplantation.

FIG. 20A: CD34⁺, CD45⁺ and CD41⁺ human cells were detected using flowcytometry and monoclonal antibodies, n=12, 16 and 8 mice, respectively.

FIG. 20B: Quantitative real time PCR using human TNFα as the probe todetect human DNA in the mouse bone marrow. Sensitivity limit was 10%.n=12, 16 and 8 mice respectively. Asterisks denote significantdifferences. Lines represent mean values.

FIG. 21A-21B: Pre-cultured CD34⁺ cells expanded with ARP₂₆ and improveplatelet counts.

FIG. 21A: 100,000 human CD34+ cells were injected together with1-200,000 CD34+ cells cultured for 10 days with no supplement (control),2 nM ARP₂₆, 2 nM ASP₄₀ or human TPO/SCF (T/S). Antibodies to humanCD45⁺, CD34⁺ and CD41⁺ were used to quantify engraftment in BM (n=6).

FIG. 21B: Human platelets per mL of mouse blood were quantified usinganti CD41, specific for human platelets. The mean differences betweengroups were large (denoted by lines). n=6.

Abbreviations: ctrl., control; T/S, TPO/SCF; po., post; wk., weeks; BM,bone marrow; PB, peripheral blood.

DETAILED DESCRIPTION OF THE INVENTION

The inventors originally described the ARP peptide as a peptide capableof inducing stem cell survival and expansion. In addition, ARP was shownto be capable of promoting myeloid and megakaryocytic differentiation[IL 130224, Inventors' co-pending US Patent Application 2003-0036632,Grisaru (2001) id ibid.].

In the present invention, the inventors demonstrate that overproductionand C-terminal cleavage of the stress-induced AChE-R isoform inducedgranulocytosis.

Effective growth and expansion of any defined hematopoietic cellpopulation involve three milestones: the first, survival of stem cells,the second, proliferation of lineage-committed progenitor cells, and thethird, expansion and maturation of terminally differentiated cells. Theexpansion of terminally-differentiated functionally-specific progenyrequires sufficient progenitor proliferation prior to maturation, whichdepends on the size of the stem cell pool. There are growth factors orcytokines that function exclusively on each one of the levels mentionedabove. For example, the stem cell factor (SCF) protects stem cells fromapoptosis and supports their survival. Alone SCF does not cause theproliferation of stem or progenitor cells. Most of the clinically usedhematopoietic cytokines drive proliferation of lineage committedprogenitors such as G-CSF, GM-CSF, erythropoietin and thrombopoietin,and work in synergy with SCF, in vitro. The ideal growth factor would bea molecule capable of maintaining the survival of stem cells and withactivity for the stimulation of committed progenitor proliferation of atleast one or more lineage, while also being capable of deriving terminaldifferentiation. The results described in the present invention defineARP as such a growth and differentiation factor, being able to supportsurvival of hematopoietic stem cells, as previously described [IL130224, Inventors' co-pending US Patent Application 2003-0036632,Grisaru (2001) id ibid.], while driving proliferation of myeloid cellsand inducing their terminal differentiation, specifically of thegranulocytic lineage, and particularly neutrophils.

Thus, it is an object of the present invention to provide the use of anAChE-R-derived peptide as an inducer of granulocytopoiesis. Yet anotherobject of the invention is to provide methods and compositions for theprevention and/or treatment of conditions leading to low white bloodcell count in general, and particularly leucopenia, and moreparticularly neutropenia. In addition, such treatments may increasecytokine production in patients who have lost the capacity to induce thesame in response to external stimuli. These include, for example, agedpatients in whom cytokine levels cannot be induced anymore because theircholinergic control over such cell population is desensitized. These andother objects of the present invention will become apparent as thedescription proceeds.

The peptide used by the invention comprises the following amino acidsequence:

(SEQ ID NO: 1) N′-GMQGPAGSGWEEGSGSPPGVTPLFSP-C′.

Said peptide is also denoted herein as ARP, or ARP₂₆.

The peptide of the invention may be isolated as a cleavage product ofAChE-R. Alternatively, the peptide is a synthetic peptide, synthesizedthrough the means of producing synthetic peptides known in the art.

Any functional derivatives and functional fragments of the above-definedpeptide may be used in the invention. The terms functional derivativesand functional fragments used herein mean the peptide, or any fragmentthereof, with any insertions, deletions, substitutions andmodifications, which is capable of inducing granulocyte celldifferentiation and/or cytokine production, particularly TPO andpro-inflammatory cytokines like TNFα, IL-6 and IL-1β.

Further, the peptide of the invention may be extended at the N-terminusand/or C-terminus with various identical or different amino acidresidues. As an example for such extension, the peptide may be extendedat the N-terminus and/or C-terminus thereof with identical or differentamino acid residue/s which may be naturally occurring or synthetic aminoacid residue/s. One example for a synthetic amino acid residue isD-alanine. An additional example for such an extension may be providedby peptides extended both at the N-terminus and/or C-terminus with acysteine residue.

Another example may be the incorporation of an N-terminallysyl-palmitoyl tail, the lysine serving as linker and the palmitic acidas a hydrophobic anchor.

In addition, the peptide may be extended by aromatic amino acidresidue/s, which may be naturally occurring or synthetic amino acidresidue/s. A preferred aromatic amino acid residue may be tryptophan.Alternatively, the peptide can be extended at the N-terminus and/orC-terminus thereof with amino acids present in corresponding positionsof the amino acid sequence of the naturally occurring C-terminal regionof AChE-R.

Nonetheless, according to the invention, the peptide to be used in theinvention may be extended at the N-terminus and/or C-terminus thereofwith various identical or different organic moieties which are notnaturally occurring or synthetic amino acids. As an example for suchextension, the peptide may be extended at the N-terminus and/orC-terminus thereof with an N-acetyl group.

The lack of structure of linear peptides renders them vulnerable toproteases in human serum and acts to reduce their affinity for targetsites, because only few of the possible conformations may be active.Therefore, it is desirable to optimize the peptide structure, forexample by creating different derivatives of the peptide of theinvention. In order to improve peptide structure, the peptide of theinvention can be coupled through its N-terminus to a lauryl-cysteine(LC) residue and/or through its C-terminus to a cysteine (C) residue.

The peptide of the invention, as well as derivatives thereof may bepositively charged, negatively charged or neutral and may be in the formof a dimer, a multimer or in a constrained conformation. A constrainedconformation can be attained by internal bridges, short-rangecyclizations, extension or other chemical modification.

The inventors have demonstrated, in the following Examples, howperipheral cholinergic stress responses, in particular overproductionand C-terminal cleavage of the stress-induced AChE-R variant, resultedin long-lasting granulocytosis, likely independent of elevated cortisollevels.

The presence of a functional glucocorticoid response element in theupstream ACHE promoter [Grisaru et al. (2001) id ibid.], combined withthe transient post-partum increase in serum cortisol [Mastorakos, G. andIlias, I. (2000) Ann. NY Acad. Sci. 900: 95-106] could explain theinitial transcriptional enhancement of ACHE gene expression inhematopoietic cells. However, the transient nature of cortisol elevationalso implies that a different transcriptional enhancing signal(s) shouldextend this response after the first few hours. That ARP₂₆ by itselfelevated ACHE gene expression in CD34+ progenitors provided a tentativeexplanation for this prolonged induction, suggesting that theoverproduced cleavable AChE-R can regulate its own production. Theseresults suggest that ARP may be used, in vivo and in vitro, for theinduction of AChE-R expression, or for re-adjusting the ratio betweenAChE-S and AChE-R.

The dose-dependent pattern of this effect further indicates that eithertoo high or too low concentrations of ARP₂₆ fail to induce AChE-R mRNAaccumulation, suggesting strict dependence of the splice shift processon previously produced AChE-R amounts which, in turn, reflects splicingregulation of the pre-AChE mRNA transcript in hematopoietic cells.ASP₄₀, the C-terminal peptide of AChE-S (denoted by SEQ ID NO:2), failedto induce such effects (FIG. 11D-11E), supporting the specificity of theeffect of ARP on prolonged granulocytosis. Vis-á-vis the resultsobtained in Example 12, ARP may be used to treat hematopoietic stemcells ex vivo, driving the cells to the granulocytic differentiationpathway.

In addition, the present findings demonstrate increased thrombopoiesisin response to the stress-induced AChE-R protein and attribute part ofthe thrombopoietic process to ARP, and to its interaction with thescaffold protein RACK1 and PKCε. This has allowed the inventors toextend the concept of what has been defined by others as “Theinflammatory reflex” [Tracey (2002) id ibid.] to the realm ofthrombopoiesis.

The effects exerted by AChE-R on the proliferation and maturation ofgranulocytes could be due to both the catalytic and the non-catalyticproperties of AChE-R as well as to the function(s) of its cleavableC-terminal peptide, ARP. The stable AChE hydrolytic activity throughoutthe peri-partum period, together with the increased AChE-R content ingranulocytes point to the possibility that granulocytes may be thesource of soluble blood AChE. This idea is reinforced by the presence ofAChE-R in the serum of peri-partum women (FIG. 9A-9B). At the catalyticlevel, AChE-R excess should lead to reduced ACh concentrations in thepost-partum serum. This, in turn, would alleviate the control overmacrophage production of pro-inflammatory cytokines, increasing theconcentration of such cytokines and inducing further proliferative andcell activation signals [Borovikova, L. V. et al. (2000) Nature 405:458-462; Tracey, K. J. (2002) Nature 420: 853-859; Wang, H. et al.(2003) Nature 421: 384-388]. The existence of nicotinic [Wang (2003) idibid.] and muscarinic [Hellstrom-Lindahl, E., and Nordberg, A. (1996) J.Neuroimmunol. 68:139-144; Mita, Y. et al. (1996) Eur. J. Pharmacol. 297:121-127]. ACh receptors on myeloid cells suggests reduced cholinergicinput to those cells as well, when under stress. Others report no directcholinergic effects on peripheral blood cells [Tracey (2002) id ibid.].However, the current study shows such effects for ARP₂₆, thus addingAChE-R production following transient increases in cortisol, and thereduced anti-inflammatory action of ACh, as additional steps to thepathway leading to protracted post-stress granulocytosis.

At the non-catalytic level, the present findings suggest the inductionof signal transduction processes by the C-terminal peptide cleaved fromAChE-R, likely through its interaction with PKCβII and its scaffoldprotein RACK1 [Inventors' co-pending US Patent Application2003-0036632]. The reported involvement of PKC signaling in myeloid cellactivation [Bassini, A. et al. (1999) Blood 93: 1178-1188] potentiallyimplicates this interaction in the maturation and/or activation ofgranulocytes in the post-partum blood.

Interestingly, the inventors have shown that, during human fetaldevelopment, AChE-R mRNA expression was observed only in the developingliver for a limited time window (FIG. 7). The transient increase inAChE-R mRNA paralleled the period of fetal liver myelopoiesis,supporting the notion that AChE-R is physiologically relevant for invivo myelopoiesis.

The induced AChE-R excess (Example 7) might be perceived as an adaptiveresponse, facilitating the production of pro-inflammatory cytokines toprotect the body from post-partum conditions, such as infections. Thisassumption is further supported by the increased production ofpro-inflammatory cytokines by mononuclear cells in the presence of thesynthetic peptide ARP₂₆. The question emerges, therefore, whichsignal(s) terminates this granulocytosis response. Because of thecircular nature of the proposed cascade process, it might be terminatedeither at the periphery or in the brain, highlighting the closeinter-relationships characteristic of long-lasting mammalian stressresponses [Kiecolt-Glaser, J. K. et al. (2003) Proc. Natl. Acad. Sci.USA 100: 9090-9095]. It is tempting to speculate that, similarly to whathappens within the central nervous system, chronically elevated AChEinduces a secondary feedback response of excess ACh production in theperiphery [Erb, C. et al. (2001) J. Neurochem. 77: 638-646]. Re-balancedACh levels can then suppress the production of pro-inflammatorycytokines in macrophages [Tracey (2002) id ibid.], terminating thegranulocytosis process. IL-1β was shown to induce ACHE gene expressionin phaeochromocytoma cells [Li, Y. et al. (2000) J. Neurosci. 20:149-155], suggesting that reduced IL-1β levels could reciprocallydecrease AChE-R (and, therefore, its cleavage product) levels back tonormal, retrieving peripheral cholinergic homeostasis.

Thus, essentially, the ARP peptide may be used as an inducer ofpro-inflammatory cytokines, particularly TNFα, IL-6 and IL-1β.

The development of transgenic mice overexpressing AChE-R allowed theinventors to further comprehend the cholinergic effect on theinflammatory response. The presence of AChE-R at high levels apparentlydoes not affect the basal status of the hematopoietic system. However itserves as an enhancer to rapid recovery of the system following aninflammatory challenge.

At least two mechanisms, not necessarily mutually exclusive, could beimplicated in that. One through the induction of pro-inflammatorycytokines, as mentioned above. Two through the induction of the putativeoncogene Spi-1 (PU.1) protein product, a hematopoietic-specific Etsfactor essential for myeloid and lymphocyte development, which has alsobeen implicated in LPS-induced signaling [Busslinger, M. (2004) Ann.Rev. Immunol. 22: 55-79]. As shown in Example 16 below (and FIG.13A-13B), PU.1 was over-expressed in bone marrow of TgR, as compared tostrain matched FVB/N mice, and probably explains why, when exposed tomild inflammatory stress, TgR mice WBC counts recovered faster thanFVB/N's, which was attributed to steady levels of granulocytes that werealtered by the insult, as well as to an exceptional recovery ofmonocytes in TgR peripheral blood, likely due to the high PU.1 levelsthat directed hematopoietic progenitors towards the myeloid lineage.Moreover, AChE-R-induced PU.1 over-expression may also provide apotential mechanism for the prolonged parturition-associatedleukocytosis.

Therefore, at the molecular level, ARP, functional fragments orderivatives thereof of compositions comprising the same, may be used forinducing the expression of PU.1. At a physiological level, ARP may beused for boosting the hematopoietic system post-partum. In events ofpost-partum hemorrhage, for example, where the mother's body losesimmense amounts of blood, while still needing to function properly inorder to care for the newborn baby, ARP may be used for hematopoieticrecovery.

In addition, the present findings point at a previously unperceivedoption for ex vivo augmentation of post-transplantation thrombopoiesis.

TPO levels are tightly controlled under normal conditions, and increaseonly when megakaryocyte and platelet production is needed. The currentstudy found a significant increase in TPO levels as well as higherplatelet counts in TgR mice over-expressing the stress-induced AChE-Rsplice variant, as compared to the strain matched FVB/N mice. LPSadministration induced a rapid fall in platelet counts in both TgR andFVB/N control mice, however, platelet recovery was considerably fasterin TgR mice than in strain-matched controls. Moreover, TgR mice showedfaster WBC recovery than controls following LPS-induced inflammation andmaintained normal RBC values while control FVB/N mice becamepancytopenic for at least 72 hrs post-LPS injection. These differencesmay be attributed to the augmented capacity of TgR BM progenitors toproliferate and differentiate into pluripotent CFU-GEMM, CFU-GM, andCFU-Mk. Although the inventors' previous reports had indicated theconnection between ARP and the megakaryocytic lineage [IL 130224,Inventors' co-pending US Patent Application 2003-0036632, Grisaru (2001)id ibid.], it was not predictable that this connection could also beattributed to the capacity of ARP to enhance TPO circulating levels, asdemonstrated in the present invention.

In principle, the AChE-R effect is double bladed. First, it can reduceACh levels, that way maintaining the production of pro-inflammatorycytokines with growth factor capacities in response to inflammatorysignals. Second, it interacts intracellularly with partner proteins,inducing signal transduction pathways and promoting cell proliferation,likely through the AChE-R partner protein RACK1 binding to PKC ε [Perry(2004) id ibid.]. PKC ε was shown to induce megakaryocyticdifferentiation in HEL and K562 cells [Racke (2001) id ibid.] and inprimary human hematopoietic stem cells [Oshevski S. et al. (1999)Biochem. Biophys. Res. Commun. 263:603-609; Marchisio M. et al. (1999)Anat. Rec. 255:7-14]. Moreover, TPO increases PKC ε expression in mousemegakaryocytes [Rojnuckarin P. et al. (2001) J. Biol. Chem.276:41014-41022], whereas blocking PKC activation inhibits plateletformation [Rojnuckarin P. et al. (2001) Blood 97:154-161].

Thus, the elevated levels of AChE-R and PKC ε in megakaryocytes from TgRmice as well as the higher plasma TPO levels in these mice supports thenotion of a cholinergic promotion of thrombopoietic signal transductionboth through the hydrolytic and the non-enzymatic features of AChE-R,involving two signaling pathways, which may engage PKCε as well.

The present findings give support to ARP as an inducer of TPO. Theinventors had previously described how ARP was able to induce CD34+ cellexpansion in combination with various growth factors, particularlyGM-CSF and TPO [Deutsch et al. (2002) id ibid.]. However, it was norclear at the time that ARP by itself could induce the expression of TPO,and increase the levels of circulating TPO. In addition, the presentinvention shows the effect of ARP on peripheral blood mononuclear cellsfrom adults, besides its influence on CD34+ populations.

Lack of proliferating megakaryocytic progenitors in BM grafts,allo-immunization and refractoriness to platelet transfusions impederecovery of patients with severe thrombocytopenia post bone marrowtransplantation. Unfortunately, TPO, the physiological regulator ofthrombopoiesis has not been clinically effective due to the paucity ofmegakaryocyte progenitors in the grafts [Kanamaru (2000) id ibid.].MGDF, the pegylated form of TPO, was retracted due to immunogenicity inhealthy donors who developed anti-TPO antibodies and became severelythrombocytopenic [Basser R. L. et al. (2002) Blood 99:2599-2602].

Thus, it is very desirable to find a drug that may more effectivelysupply TPO for these patients. Hence, administration of atherapeutically effective amount of ARP, its functional fragments orderivatives, or compositions comprising thereof, may be one way ofinducing TPO production and consequently increasing the number ofmegakaryocytic progenitors and platelets.

As mentioned throughout the present specification, said therapeuticeffective amount, or dosage, is dependent on severity and responsivenessof the disease state to be treated, with the course of treatment lastingfrom several days to several months, or until a cure is effected or adiminution of the disease state is achieved. The therapeutic effectivedosage may be determined by various methods, including generating anempirical dose-response curve, predicting potency and efficacy of acongener by using quantitative structure activity relationships (QSAR)methods or molecular modeling, and other methods used in thepharmaceutical sciences. Optimal dosing schedules may also be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. In general, the dosage may be givenonce or more daily, weekly, or monthly. Persons of ordinary skill in theart can easily estimate repetition rates for dosing based on thepatient's response to the active agent.

Increasing the numbers of megakaryocyte precursors in a graft ofhematopoietic precursor cells should shorten the extended period ofsevere thrombocytopenia, promoting successful engraftment of long termrepopulation of stem cells, the appropriate targets for endogenous orexogenous TPO.

In a further aspect, the present invention provides the use of anAChE-derived peptide or its functional fragments or derivatives, in thepreparation of a pharmaceutical composition for any one of the treatmentand/or prevention of conditions that trigger low granulocyte count, suchas leucopenia, and particularly neutropenia, and in pre-transplantpriming of hematopoietic stem cells, wherein said peptide is denoted bySEQ ID NO:1.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g., GennaroA. R. ed. (1990) Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., and especially pages 1521-1712 therein.Essentially the preparation of compositions involves admixing the ARPpeptide with pharmaceutically acceptable carriers, diluents orexcipients, and further optionally with desirable additives.

Blood cell inflammatory and immune processes involve a finely tunedbalance between myeloid cell activation, proliferation anddifferentiation. Reduced AChE-R densities on the cell surface of Blymphocytes under stress should further increase the chances of ACh toactivate these cells by interacting with their ACh receptors [Wang(2003) id ibid.]. The development and stress-induced changes in ACHEgene expression of myeloid cells are hence likely to facilitate thehematopoietic responses to external stimuli.

In an even further aspect, the present invention provides a method oftreatment of conditions that induce leucopenia, comprising the steps ofadministering a therapeutically-effective amount of an AChE-derivedpeptide or a composition thereof to a subject in need, wherein saidAChE-derived peptide is denoted by SEQ ID NO:1. Leucopenia includes anycondition in which the number of white blood cells is reduced. Oneparticular condition is neutropenia.

As mentioned herein, administration of the peptide of the invention, itsfunctional fragments or derivatives, or compositions comprising thereof,is preferably via intravenous. Administration directly into the bonemarrow cavity may also be advisable, in order to maximize the contactbetween ARP and hematopoietic progenitors. Intraperitoneal andintradermal administrations may also be comtemplated.

Thus, the invention also refers to an in vivo method for the preventionand/or treatment of conditions wherein lymphocyte activity is reduced,such as chronic stress, autoimmune diseases, inflammation, rheumatoidarthritis, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),fibromyalgia, multiple chemical sensitivity, post-irradiation,chemotherapy in a subject in need, comprising administering atherapeutically-effective amount of an AChE-derived peptide, itsfunctional fragments or derivatives, or compositions comprising thereof,to an individual suffering or prone to said conditions, wherein saidpeptide is denoted by SEQ ID NO:1.

Said method may also be accomplished in vitro or ex vivo, similarly towhat is described below, through admixing isolated immature blood cells(preferably enriched for the CD34+ population), with ARP for apre-determined amount of time, which should be sufficient for increasingthe number of committed hematopoietic progenitor cells, and especiallyfor cells of the granulocytic and megakaryocytic lineages.

As used herein in the specification and in the claims section below, theterm “treat” or treating and their derivatives includes substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical symptoms of a condition orsubstantially preventing the appearance of clinical symptoms of acondition.

As shown in Example 5, B lymphocytes in post-delivery mothers lose mostof their surface AChE-R, but maintain high levels of cytoplasmic AChE-Rexpression, an unprecedented response pattern unique to these cells.This result suggests that the changing pattern of AChE molecules inlymphocytes might reflect a change in lymphocytic activity in responseto variations in cholinergic stimuli, under stress situations. Thus, theAChE peptide might be a potent regulator of lymphocytes activity, invivo, ex vivo and in vitro.

In addition, the invention refers to a method for inducing a shift inthe activity of lymphocytes in vitro or ex vivo, comprising contactingan AChE-derived peptide with lymphocytes for a suitable period of time.

Hence also, the present invention discloses a method for detectingchanges in the activity of lymphocytes, comprising measuring theexpression of AChE-R on the surface of lymphocytes.

As shown in Example 23, priming CB CD34⁺ cells with ARP₂₆ increasedsignificantly the number of human CD45⁺ cells found in the mouse BM sixweeks post transplant. Quantitative PCR analysis confirmed largercontent of the human TNFα gene (as a marker for human-originating cells)in mice transplanted with ARP₂₆-primed cells. Additionally, incubatingCB CD34⁺ cells for 10 days with 2 nM ARP₂₆ improved the recovery fromthrombocytopenia in NOD/SCID mice. As shown previously, CD34⁺ cellsplaced in culture usually loose their ability for long-term engraft dueto differentiation and commitment manifested by the acquisition of theCD38 marker [Guenechea (1999) id ibid.; Li K. (1999) id ibid.].Nevertheless, these cells produce more AChE-R [Grisaru (2001) id ibid.]and can hence support megakaryopoiesis when mixed with immature CD34⁺providing a clear engraftment advantage of CD45⁺, CD34⁺ and CD41⁺megakaryocyte human cells. The current study proposes a novel strategyto facilitate thrombopoiesis, which involves exposing stem cells to ARP,its functional fragments or derivatives, or a composition comprisingthereof, for a pre-determined period of time sufficient for increasingthe number of granulocytic and megakaryocytic progenitors. This exposuremay be in vivo, through administration of the peptide to a subject inneed, or in vitro/ex vivo. When in vitro/ex vivo said exposure involvesobtaining hematopoietic cell precursors and admixing said precursorcells with ARP, at concentrations in a range between 0.2 nM up to 100 nMof ARP, preferably between 1 nM and 20 nM of ARP, for a period ofbetween at least 24 hours up to 15 days. Exposed cells may be recoveredafter 3, 6, 8, 10 or 12 days of incubation with the ARP peptide, itsfunctional fragments or derivatives or compositions comprising thereof.The exposure may be performed by culturing said cells in the presence ofARP. Said treatment aims at improving stem cell engraftment andshortening post-transplant thrombocytopenia.

In addition, the above treatment, or method of priming of hematopoieticstem cells pre-transplant, is also useful for treating a subject in needof granulocytes.

It should be noted that such a pre-transplant priming method may beperformed in cells from the subject in need of said transplant, or incells from another subject, preferably immunocompatible with the host.Thus, the pre-transplant priming may be performed in autologoustransplantations or in allogeneic transplantations. In case ofallogeneic transplantations, adequate immunocompatible matchinghost-donor pairs shall be evaluated by the medical professional in careof the patient.

Lastly, the invention also provides an ex vivo method of inducing adultblood cells to produce cytokines, comprising obtaining said cells from asubject in need of cytokine-producing blood cells, isolating immaturecells and contacting said cells with an AChE-derived peptide, whereinsaid peptide is denoted by SEQ ID NO:1.

Thus, cells may be treated as described above, by admixing with orculturing in the presence of ARP, its functional fragments orcompositions comprising thereof, but now with the goal of inducing theproduction of cytokines. These may be, for example, TPO, orpro-inflammatory cytokines, such as TNFα, IL-6 or IL-1β.

This method is particularly advantageous for patients with neutropenia.

Neutropenia is a decrease in circulating neutrophils in the peripheralblood. The absolute neutrophil count (ANC) defines neutropenia. ANC isfound by multiplying the percentage of bands and neutrophils on adifferential by the total white blood cell count. An abnormal ANC isfewer than 1500 cells per mm³. Neutropenia can be present (though it isrelatively uncommon) in normal healthy individuals, notably in blacksand Yemenite Jews.

Causes of neutropenia from disease can be categorized as resulting fromdecreased production of white blood cells, destruction of white bloodcells after they are produced, or pooling of white blood cells(accumulation of the white blood cells out of the circulation).

Diseases causing decreased production of white blood cells include drugtoxicity, vitamin deficiencies, and medical diseases such as blooddiseases, infections (virus diseases, tuberculosis, typhoid),abnormalities of the bone marrow disorders, or be cyclic (varying inseverity week to week, month to month, perhaps related to biorhythms).Several leukemias may also result in neutropenia. Destruction of whiteblood cells can occur as a result of antibodies attacking the cells(such as in Felty's syndrome) or from drugs stimulating the immunesystem to attack the cells.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures Tissue and Cell Preparations:

-   -   Cord Blood (CB) cells were retrieved from umbilical cords of        newborns of uncomplicated full-term pregnancies, as described        [Grisaru (2001) id ibid.], in anti-coagulant citrate dextrose        solution formula A-supplemented bags (Baxter, Deerfield, Ill.).    -   Peripheral blood from adult healthy women and from mothers        within the first 24 hours post-delivery was obtained from        discarded samples of routine blood examinations.    -   Only healthy, medication-free patients and neonates and only        pregnancies which were uneventful up to term were included in        this study.    -   Paraffin-embedded sections from electively aborted normal human        embryos were prepared as previously described [Grisaru (1999b)        id ibid.].    -   Peripheral mononuclear and CD34+ cells were enriched to 85% by        separation on gelatin and Ficol-Hypaque gradients followed by        CD34 immune magnetic beads (Dynal, Great Neck, N.Y.),        essentially as described [Grisaru (2001) id ibid.; Pick, M. et        al. (1998) Br. J. Haematol. 103:639-650]. Alternatively, CD34⁺        stem cells were purified using a CD34⁺ progenitor cell isolation        kit (PE, Miltenyi Biotec GmbH, Gladbach, Germany), according to        manufacturer's instructions.

The use of human material in this study was approved by the Tel AvivSourasky Medical Center Ethics Committee according to the regulations ofthe Helsinki accords.

Animal Models:

Transgenic Mice

All animal experiments were approved by the animal ethics committee ofThe Hebrew University. Transgenic (TgR) mice expressing human AChE-Rwere generated by injecting a DNA construct including the proximal CMVpromoter-enhancer followed by exons 2, 3, 4, pseudointron 4′ and exon 5of the human ACHE gene (GenBank Accession No. M55040) and an SV40polyadenylation signal, into fertilized eggs of FVB/N mice [Sternfeld etal. (1998b) J. Neurosci. 18: 1240-1249]. This transgene presentedunimpaired mendelian inheritance over 5 generations [Sternfeld, M. etal. (1998a) J. Physiol. Paris 92: 249-255].

To generate acute inflammation, 5 μg LPS of E. coli origin (Sigma, StLouis, Mich.) was injected intraperitoneally (IP) in 400 μl of phosphatebuffered saline (PBS, Biological Industries, Beth Haemek, Israel).Peripheral blood was drawn from the retroorbital vein of TgS and FVB/Nmice, collected in EDTA (7.5%) tubes. Marrow cells were harvested fromthe mouse femur bones with a 26 G needle pre-washed with heparin, andkept in phosphate-buffered saline (PBS).

NOD/SCID mice: Non-obese diabetic SCID (NOD/SCID) mice were maintainedunder defined flora conditions in the animal facility at the WeizmannInstitute of Science (Rehovot, Israel) in sterile intra-ventilated cages(IVC; Techniplast, Buguggiate, Italy). Mice were sub-lethally irradiatedwith 375 cGy at 67 cGy/min from a ⁶⁰Co source and 24 hrs later weretransplanted with 100,000 human cord blood CD34⁺ cells by intravenousinjection in 400 μl of Hank's Balanced Salt solution (HBSS, BiologicalIndustries, Beit Haemek, Israel). Mice were sacrificed between 2 and 6weeks post transplant, samples of PB (orbital bleed) and BM (femur bone)were removed and human engraftment assessed.

Variant-Specific Antibodies

Monoclonal human antibody fragments were selected from a phage displaylibrary, using ASP, a synthetic peptide with the C-terminal sequenceunique to human AChE-S, as target for selection. The 90% pure anti-ASP1antibody was obtained as soluble single-chain Fv (scFv) including a myctag and a His6 tail [Flores-Flores, C. et al. (2002) J. Neural Transm.62(suppl):165-179]. Polyclonal affinity-purified rabbit antibodiesdirected towards the C-terminal sequence unique to human AChE-R (ARP)were obtained after repeated rabbit challenges with a glutathioneS-transferase-ARP fusion protein (FIG. 1) [Sternfeld, M. et al. (2000)Proc. Natl. Acad. Sci. U.S.A. 97:8647-52].

Detecting AChE Variants

2×10⁶ cells (50 μl) were incubated (30 minutes, 4° C.) withanti-CD45-PerCP (20 μl, 0.4 μg; BD Bioscience, San Jose, Calif.) andscFv purified anti-ASP1 (5 μl, 1 μg) or rabbit anti-human polyclonalanti-ARP (5 μl, 1.4 μg), washed with 15 ml 1% BSA in phosphate-bufferedsaline (PBS), and centrifuged (600×g, 5 minutes, 4° C.). Secondaryantibodies were added to 50 μl of resuspended cells (30 minutes, 4° C.),anti-c-myc FITC for detecting anti-ASP (5 μl, 1 μg; Caltag, Burlingame,Calif.) or anti-rabbit-FITC for detecting anti-ARP (3 μl, 3 μg; Zymed,San Francisco, Calif.). Cells were washed as above, and red blood cellswere lysed with 1 ml of 1:10 diluted FACS lysis buffer (BD Bioscience,Palo Alto, Calif.; 12 minutes, 4° C.). Non-specific staining wasevaluated by incubating with FITC-labeled secondary antibodies andanti-CD45-PerCP only. Surface AChE-R expression was detected onlymphocytes by double-staining with anti-CD19-APC (5 μl, 1 μg; Caltag)to identify B cells or anti-CD3-APC (5 μl, 1 μg; Caltag) to identify Tcells. Intracellular proteins were detected in permeabilized cells(IntraStain, Dako, Glostrup, Denmark). Cytochemical staining ofcatalytically active AChE was performed as previously reported[Lev-Lehman, E. et al. (1997) Blood 89:3644-53].

Flow Cytometric Immunophenotyping and AChE-R Detection

To detect AChE-R, cells were incubated with CD45-PerCP (BD Bioscience,Palo Alto, Calif.), followed by permeabilization using the IntrastainKit (Dako, Glostrup, Denmark), staining with rabbit anti-human ARP₂₆antibodies [Sternfeld, M. et al. (2000) id ibid.], and detection withFITC-conjugated goat anti-rabbit Fab antibody (Jackson Laboratory, BarHarbor, Me.). Mean fluorescence intensity (MFI) served as a measure ofAChE-R content in analyzed cells. When multiplied by the percentfractions of AChE-R-positive cells, the MFI values reflected the totalcontent of AChE-R in the analyzed blood cell samples. Myeloid markers ofmaternal blood cells were analyzed by the following combination ofmonoclonal antibodies: anti-CD15-FITC (Dako, Glostrup Denmark),anti-CD33-PE (BD Bioscience, Palo Alto, Calif.), anti-CD45-PerCP (BDBioscience, Palo Alto, Calif.) and anti-CD14-APC (Caltag, Burlingame,Calif.). Corresponding MFI values reflected the amount of receptor onthe surface of granulocytes and monocytes. Expanded CD34+ cells wereanalyzed by 4-color flow cytometry with FITC-conjugated anti-CD15 andPE-conjugated anti-CD33, PerCP-conjugated anti-CD34, and APC-conjugatedanti-CD38 (all antibodies purchased from BD Bioscience, Palo Alto,Calif.) using a FACSCalibur with CellQuest software (BD Bioscience, PaloAlto, Calif.). Relevant isotype control antibodies were used to detectnon-specific background fluorescence. The total number of expanded cellsfor each lineage was calculated by multiplying their relativeproportions by the number of viable cells in each culture.

Immunophenotyping of hematopoietic population in mouse bone marrow (BM)and peripheral blood (PB) used the following antibody panels: 1)Gr.1-FITC (clone RB6-8C5, Caltag Laboratories, Burlingame, Calif.),CD11b-PE or -APC (clone M1/70.15, Caltag), CD45-TC (Clone YW62.3,Caltag) to detect the myeloid lineage; 2) CD19-FITC (Clone 6D5, Caltag),CD4-PE (Clone CT-CD4, Caltag), CD3-APC (Clone CT-CD3, Caltag) to detectthe lymphoid lineage.

Solubilization of Cellular Antigens:

Cord red blood cells were isolated by centrifugation (600×g, 20minutes). The plasma and upper layer of cell sediment were removed.Leukocytes were isolated on Ficoll-Hypaque (Pharmacia, Peapack, N.J.).Granulocytes found below the Ficoll-Hypaque layer were isolated andremaining red blood cells were lysed (BD Bioscience, Palo Alto, Calif.).Mononuclear cells found above the Ficoll-Hypaque, containing monocytesand lymphocytes were washed in 1% BSA-PBS (600×g, 5 minutes, 4° C.),re-suspended in 10 ml of Iscove's modified Dulbecco medium (IMDM,Biological Industries, Beit Haemek, Israel) supplemented with 10% fetalcalf serum (Biological Industries, Beit Haemek, Israel) and incubated(90 minutes, 37° C., 100% humidity, 5% CO₂), allowing monocytes toadhere. Non-adherent cells containing highly enriched lymphocytes werewashed with 1% BSA-PBS and adherent monocytes were scraped and washed in1% BSA-PBS. Cell populations, all above 95% pure (tested with antibodiesspecific for the population and flow cytometry), were washed with PBSand re-suspended in high salt detergent buffer (300 mM NaCl, 0.5% TritonX-100, 50 mM Tris HCl, pH 7.6), including the protease inhibitorcocktail Complete Mini (Roche Molecular Biochemicals, Mannheim,Germany). After 1 hour shaking at 4° C., samples were centrifuged (10minutes, 10,000×g, 4° C.). Supernatants were stored at −80° C. forfurther analysis. Protein concentration was determined using a Lowryassay kit with albumin as protein standard (Bio-Rad, Hercules, Calif.).

Immunoblots:

Anti-ASP1 antibody displayed on the phage surface [Flores-Flores (2002)id ibid.] was used. The pellet of separated cells was resuspended in 10ml of denaturing buffer (2% SDS, 50 mM Tris HCl, pH 6.8). Soluble celllysates (6 μg of protein) or plasma samples (to detect AChE-R incirculation, total of 20 μg protein) were run on 4-20% polyacrylamidegels and electroblotted. Membranes were blocked (10% BSA, PBS, 0.5%Tween 20, 18 hours, 4° C.), and incubated with the phage carrying theanti-ASP1 antibody (2.6×10⁸ transforming units/ml, blocking buffer, 2 hat room temp.) or anti-human ARP₂₆ antibodies [Sternfeld (2000) idibid.]. Following washes (PBS-0.5% Tween20), membranes were incubatedwith horseradish peroxidase/anti-M13-conjugated or anti-rabbit antibody(Amersham Pharmacia Biotech, Little Chalfont, UK) for 1 hour at roomtemperature, and diluted 1:10000 in 5% BSA-PBS, 0.1% Tween20. Peroxidaseactivity was detected using an ECL kit from Amersham. Blots wereanalyzed using the luminescence tool of Adobe Photoshop 7.0 ME (AdobeSystems, Inc., San Jose, Calif.).

Surface Plasmon Resonance (SPR):

SPR measurements (BIAcore 3000 System, Uppsala, Sweden) used theanti-ASP1 scFv and anti-ARP antibodies immobilized on a CM5 sensor chipthrough their primary amine groups [Johnsson, B. et al. (1991) Anal.Biochem. 198:268-77]. The matrix was activated with 70 μl of 0.4 MN-ethyl-N′-(dimethyl-aminopropyl)-carbodiimide and 0.1 MN-hydroxysuccinimide, and 200 μg/ml of the particular antibody in 10 mMsodium acetate, pH 3.5, were injected at a flow rate of 10 μl/min in 10mM HEPES, pH 4.0, 150 mM NaCl, 3.4 mM EDTA and 0.005% polysorbate 20 toreach surface density of between 3000 to 6000 resonance units (RU).Remaining activated carboxyl groups were blocked by injecting 70 μl of 1M ethanolamine hydrochloride. Cord blood cell extracts in high saltdetergent buffer were brought to 0.83 mg protein/ml in 10 mM HEPES pH4.0 with 150 mM NaCl, 3.4 mM EDTA, 0.005% polysorbate 20. Carboxymethyldextran was added to avoid non-specific binding of protein to thesurface matrix. 60 μl extract doses were injected through the flowcellto which the antibody was immobilized and through a reference surface(to which no antibody was immobilized) for 2 minutes. A 10 μl pulse of 2M NaCl achieved regeneration of the antibody in the flowcell. Datamanagement involved multi-parameter Student's t-test statistics with pvalues<0.05 considered significantly different.

Cell Counts and Serum Tests

Plasma was separated from blood samples used for cell counts with theCoulter Gen-S analyzer (Beckman Coulter, Miami, Fla.). Plasma cortisollevels were measured by eletrochemiluminescence immunoassay (ECLA) andanalyzed by Elecsys 1010/2010 and modular analytics E170 (Roche,Indianapolis, Ind.). AChE activity was determined in the plasma by astandard colorimetric assay in the presence of 10⁻⁵ iso-OMPA, aselective inhibitor of butyrylcholinesterase (BChE). Mononuclear cells(2.5×10⁶ cells/mL) from healthy adult women were cultured for 24 hoursin the presence or absence of the noted peptides. The supernatant wascollected following centrifugation (4300 rpm, 10 min) and filtration(0.2 μm). Cytokine levels, including TNFα, IL-1β, IL-6, IL-10 andIL-12p-70, in the plasma and cell supernatants were assessed by flowcytometry (BD Bioscience, San Jose, Calif.) using a particle-basedimmunoassay (CBA kit, BD Bioscience, Palo Alto, Calif.). Dataacquisition and analysis utilized CellQuest and Microsoft Excel software(BD Bioscience, Palo Alto, Calif.).

In Situ Hybridization

In situ hybridization procedures were performed on freshly isolatedcells using 5′-biotinylated, 2′-O-methylated AChE cRNA probescomplementary to 3′-alternative human ACHE exons as previously described[Grisaru (2001) id ibid.]. Labeling intensity was assessed as thepercent cytoplasmic red pixels and normalized by subtraction ofbackground signals. Confocal microscopic scans of the cells wereobtained using a MRC-1024 Bio-Rad confocal microscope (Hemel HempstedHerts., UK). ANOVA and t-test were used for statistical calculations.

RT-PCR and Real Time RT-PCR

Total RNA was purified from bone marrow with the RNeasy kit (Qiagen),followed by treatment with DNase I (Qiagen) according to manufacturer'sprotocol. RNA quality was confirmed by electrophoresis on agarose gel,and analysis of OD ratios at 260 nm versus 280 nm—all values werebetween 1.8 and 2.1. cDNA was prepared from this RNA using the Improm IIkit (Promega, Madison, Wis.). For each reaction, 2.4 μl of 25 mM MgCl₂,4 μl of X5 buffer, 1 μl reverse transcriptase, 1 μl dNTP mix (10 mM ofeach), 1 μl random hexamers (of 50 μM stock, Sigma), 0.5 μl RNaseinhibitor (20U, Promega) and 2 μl sample RNA (200 ng/μl) were mixed withdiethyl pyrocarbonate (DEPC) water to a final volume of 20 μl. Thereverse transcription reaction was 45 minutes at 42° C., 5 minutes at90° C., and then the samples were left at 4° C.

Experiments with real-time quantitative PCR were performed with theLightcycler™ system (Roche, Switzerland) and SYBR Green PCR Master Mix(Applied Biosystems). Primers for Ikaros1 and mCtBP were designed usingthe Lightcycler™ sequence-detection software (Roche, Switzerland).Primer sequences for mFOG, mGATA1, Runx1/AML1, PU1, β-globin, STAT5, andthe housekeeping gene β actin (SEQ ID NOS:3-14), as well asamplification conditions, are listed in Table 1. Purity of the PCRproducts was verified by a melting curve analysis using the Lightcycler™system, and by agarose gel analysis.

TABLE 1 Primer sequences used for Real Time PCR Annealing PrimerSequence temperature GATA1 + 5′-3′ TCTTCTCTCCCACTG 65° C. (SEQ ID NO: 3)GGAGCCCT GATA1 − 5′-3′ CTTCTTGGGCCGGAT (SEQ ID NO: 4) GAGAGGCC LMO2 +5′-3′ TGGATGAGGTGCTGC 65° C. (SEQ ID NO: 5) AGATA LMO2 − 5′-3′CCCATTGATCTTGGT (SEQ ID NO: 6) CCACT RUNX1/AML1 + 5′-3′ ACTTCCTCTGCTCCG65° C. (SEQ ID NO: 7) TGCTA RUNX1/AML1 − 5′-3′ GTCCACTGTGATTTT(SEQ ID NO: 8) GATGGC PU.1 + 5′-3′ GATGGAGAAAGCCAT 55° C. (SEQ ID NO: 9)AGCGA PU.1 − 5′-3′ TTGTGCTTGGACGAG (SEQ ID NO: 10) AACTG STAT5b + 5′-3′GGGACTCAATAGATC  65° C. (SEQ ID NO: 11) TTGATAATCC STAT 5b − 5′-3′AACTGAGCTTGGATC (SEQ ID NO: 12) CGCAGGCTCT Actin + 5′-3′ CAATTCCATCATGAA65° C. (SEQ ID NO: 13) GTGTGAC Actin − 5′-3′ ATCTTGATCTTCATG(SEQ ID NO: 14)  

For quantification of transcript levels, the target concentrations atwhich each transcript was amplifying at the log linear range was tested,using serial dilutions of cDNA preparations (1:1, 1:3, 1:9, 1:81, where1:1 corresponds to a concentration of 400 ng/ul at the reaction mix).The efficiencies for all targets were very similar (amplification of ˜n^(1.8) per PCR cycle) when RT products were diluted 1:5. Amplificationreactions were performed in a final volume of 10 μl containing 1 μl of5-fold diluted RT reaction product, 1 μl SYBR Green PCR Master Mix, 10μM primer, and nuclease-free water.

Acetylthiocholine Hydrolyzing Activity (AThCh)

Mouse plasma samples were separated from the nucleated cell fraction bycentrifugation at 4300 rpm (2000×g, 20 min) sterilized through a 0.2 μmpore size filter and stored in aliquots at −70° C. until use. BM cellswere washed with PBS (Sigma) and re-suspended in low salt detergentbuffer (300 mM NaCl, 0.5% Triton X-100, 50 mM Tris HCl, pH 7.6),containing protease inhibitor cocktail (Roche Molecular Biochemicals).AThCh activity was as previously described [Kaufer (1998) id ibid.].

Ex Vivo Expansion of Hematopoietic Progenitor Cells:

For cell priming experiments 100,000 fresh CB CD34⁺ cells weresupplemented with 2 nM of peptide, ARP₂₆ [Grisaru (2001) id ibid.],ASP₄₀ [Grisaru (2001) id ibid.] or no supplement for 2 hours andinjected into mice. For 10 day cultures CB CD34⁺ cells were expanded inliquid cultures in the presence of one of the following growthsupplements: ARP₂₆ (2 nM, synthetic peptide), ASP₄₀ (2 nM, syntheticpeptide), rhu-TPO (1 ng/mL) (R&D) together with rhu-SCF (50 ng/mL;Genzyme Diagnostic, Cambridge, Mass., USA) or no supplement (control).ARP₂₆, and ASP₄₀ were synthetically produced. PBAN (a negative controlinsect peptide) [Nijholt (2003) id ibid.] were also used for cellexplansion. Liquid cultures were initiated and maintained in 24-welltissue culture plates (1×10⁵ cells/well in 1 mL). Cells were grown for10 days at 37° C. in 5% CO₂ in a fully humidified atmosphere in IMDMsupplemented with 5% autologous plasma. At 3-day intervals, cultureswere supplemented with the same growth factor(s) and cells were countedby trypan blue exclusion and diluted to maintain cultures atconcentrations no higher then 100,000 cells/mL [Pick M. et al. (2002)Exp. Hematol. 30:1079]. Cultured cells were injected into NOD/SCID miceat a concentration of 100,000 or −200,000 together with 100,000unexpanded CD34⁺ cells per mouse as indicated.

Progenitor Colony Assays

GM-CFU: mouse BM cells were cultured at 2×10⁵ cells per 35 mm tissueculture dish (Corning Inc., NY) in IMDM (Biological Industries, BeitHaemek, Israel) supplemented with 0.8% methyl cellulose (Sigma-AldrichCorp., St. Louis, Mo.), 10% FCS (Biological Industries, Beit Haemek,Israel) and 5×10⁻⁴ M 2-beta-mercaptoethanol (2-ME) (Sigma), 5 ng/mLrecombinant mouse-granulocyte macrophage—colony stimulating factor(rmo-GM-CSF, R&D Systems Inc., Minneapolis, Minn.), 10 ng/mL rmo—stemcell factor (rmo-SCF, R & D), 3 U/mL rhu-erythropoietin (rhu-EPO, R & DSystems Inc., Minneapolis, Minn.) and rmo-Interleukin-3 (rmo-IL-3, R &D) in 5% CO₂ at 37° C. Colonies of more than 40 cells were counted atday 10.

BFU-E: 2×10⁵ BM cells per 35 mm dish were cultured in Alpha-MEM(Biological Industries, Beit Haemek, Israel) supplemented with 0.8%methyl cellulose, 10% FCS, 10% bovine serum albumin (BSA, BoehringerIngelheim GmbH, Germany) and 5×10⁻⁴ M 2-ME, 3 U/mL rhu-EPO and 10 ng/mLrmo-SCF. Red cell clusters were counted at day 12 of incubation in 5%CO₂ at 37° C.

CFU-Mk: 2×10⁵ BM cells per 35 mm dish were cultured in McCoy's Medium(Biological Industries, Beit Haemek, Israel) supplemented with 0.3% agar(Difco, Mich.), 10% FCS and 10⁻⁴ M 2-ME, 2 ng/mL rmo—thrombopoietin(rmo-TPO, R&D Systems Inc., Minneapolis, Minn.) and 10 ng/mL rmo-SCF in5% CO₂ at 37° C. for 10 days. Plates were placed into an oven for 2 hrsat 45° C. with Whatmann No. 1 filter paper discs carefully placed overthe agar layer. The filter paper was then gently removed and platesincubated with AChE substrate (10 mg acetylthiocholine iodide dissolvedin 15 mL of 0.1M dibasic sodium phosphate, 1 mL of 0.5 M sodium citrate,2 mL 30 mM cupric sulfate and 2 ml 5 mM potassium ferricyanide) for upto 24 hrs at room temperature or until colonies turned brown in color.

Quantification of Cytokine Levels

Mouse TPO, EPO, Tumor necrosis factor-alpha (TNF-α and IL-6 levels inplasma of TgR and FVB/N mice were determined using Quantikine murineenzyme-linked immunosorbent assay (ELISA) kits (R&D), according to themanufacture's instructions.

Immunhistochemistry

BM cell smears were fixed for 15′ with methanol, washed 3 times with PBSand then 3 times with 100 mM glycine to quench auto-fluorescence.Blocking buffer included 1% donkey (Santa Cruz Biotechnology Inc., SantaCruz, Ca), or 1% goat serum (Santa Cruz) for 30 min at room temperature.Antibodies against human AChE-R (Rabbit, 0.6 ug/slide) [Sternfeld M. etal. (2000) Proc. Natl. Acad. Sci. USA 97:8647-8652], PKC ε (mouse, 0.5ug/slide) (BD Biosciences, Palo Alto, Calif.) and RACK1 (mouse, 0.25ug/slide) (BD Bioscience, Palo Alto, Calif.) were incubated for 60′ withblocking buffer. TBST (Tris buffered saline with 0.2% Tween 20) was usedto wash slides after each antibody incubation. For detection,biotin-SP-conjugated AffiniPure goat anti mouse IgM or donkey antirabbit IgG (1:200, Jackson ImmunoResearch Laboratories Inc., West Grove,PN) and Cy3™ conjugated streptavidin (1:200, Jackson ImmunoResearchLaboratories Inc., West Grove, PN) were each incubated for 30 min atroom temperature. May-Grünwald staining was performed to morphologicallyidentify megakaryocytes.

Detection of Human Cells Engraftment in NOD/SCID Mice

NOD/SCID mice were sacrificed 2 to 6 weeks post-transplantation and PBand BM were analyzed following lysis of mature RBCs with FACS lysisbuffer (BD Bioscience, Palo Alto, Calif.). 5×10⁶ cells were incubatedwith human antibodies anti-CD41a-FITC (Beckman/Coulter, Fullerton,Calif.), anti-CD34-PE (BD Bioscience, Palo Alto, Calif.) and anti-CD45PerCP (BD Bioscience, Palo Alto, Calif.) (30 min, 4° C.). To followhuman platelet engraftment, PB of NOD/SCID mice was stained withanti-human CD41a-FITC and anti-mouse CD41a-PE (BD Bioscience, Palo Alto,Calif.), and a specific platelet gate was placed at acquisition.

At least 500,000 events per sample were acquired with a BD FACS Calibur(BD Bioscience, Palo Alto, Calif.). Data analysis used Cell Quest andCell Quest Pro software (BD Bioscience, Palo Alto, Calif.). Matchedisotype controls for all antibodies were used to detect backgroundfluorescence (supplied by Caltag and BD Bioscience, Palo Alto, Calif.).All human antibodies were pre-tested on naïve-untransplanted mice totest for any cross-reactivity. To detect human-originated cells, BM DNAwas extracted (QIAprep Spin Miniprep Kit, Qiagen) according tomanufacturer instructions. DNA samples (100 ng, 2 μl) were incubated in10 μl containing 1 μl Light Cycler™ DNA master hybridization probe(Roche Molecular Biochemicals), 1 μl primers (5 μM sense and 5 μMantisense), 1 μl probes (5 μM anchor and 5 μM sensor), 1.2 μl MgCl₂ (3mM) and nuclease-free water. TNFα primer and probe sequences are listedbelow in Table 2 (SEQ ID NOS:15-20). PCR involved 45 cycles (95° C. for10 sec, 65° C. for 7 sec, and 72° C. for 20 sec). Standard curves weregenerated by mixing mononuclear cells (MNCs) from human CB together withmouse BM, total number of cells being 5×10⁶ per concentration withmixtures of 0, 0.5, 1, 2, 5, 10, 20, 40, 60, 80 and 100% human cells.The human probe and primer were found negative in naïve mice.

TABLE 2 DNA sequence of primers and probes for TNFα. Name 5′-3′ sequenceSequence Name Human sense AGGAACAGCACAGGCCTTAGTG SEQ ID NO: 15 Human AAGACCCCTTCCAGATAGATGG SEQ ID NO: 16 antisense Human probeGCCCCTCCACCCATGTGCTCC- SEQ ID NO: 17 FLAC-RED640 CACCCACCACCATCAGCCGCATCSEQ ID NO: 18 Mouse sense GGCTTTCCGAATTCACTGGAC SEQ ID NO: 19 Mouse CCCCGGCCTTCCAAATAAA SEQ ID NO: 20 antisense FL-sensor, AC-.anchor*Nucleotide sequences are based on human and mouse TNF α genes (Gen BankAccession Numbers M26331 and Y00467, respectively) [Nitsche A. et al.(2001) Haematologica 86:693-699].

Example 1 Evaluating AChE Splice Variants in Hematopoietic CellPopulations

Cytochemical staining of smeared blood cell preparations revealedacetylthiocholine-hydrolysing AChE in blood cells from the newborn,adult and post-partum sources. Particularly prominent intracellularstaining was observed in adult and post-partum granulocytes, whereasenzyme activity on the cell surface was clearly observed on lymphocytes,granulocytes and monocytes from adult blood, compatible with theinventors' previous findings [Lev-Lehman (1997) id ibid.], but only ongranulocytes from post-partum mothers. FIG. 2A portrays representativemicrographs (one cell from 30 analyzed) of this staining, and FIG. 2Bincludes activity staining combined with morphology.

To attribute enzyme activities to specific AChE variants and exploretheir surface-cytoplasmic localization, flow cytometry was used, whichcombines physical characteristics of these cells with specific surfaceantigens. CD45, a glycosylated trans-membrane phosphatase which isexpressed on the membrane of granulocytes, monocytes and lymphocytes atdifferent intensities, but not in erythrocytes [Craig, W. et al (1994)Br. J. Haematol. 88:24-30; Xu, Z. and Weiss, A. (2002) Nat. Immunol. 3:764-71]. CD45 was used to identify these blood cell populations fromseveral human sources, which included adult non-pregnant women, adultwomen post-partum and cord blood from their newborns.

Antibodies directed to the unique C-terminal sequences of human AChE-S[Flores-Flores (2002) id ibid.] and AChE-R [Sternfeld (2000) id ibid.]were used in conjunction with CD45 labeling to analyze the expression ofthe corresponding variants or fragments thereof in the sub-classifiedblood cell populations. Flow cytometry measurements using naive orpermeabilized cells enabled distinction between cell surface andintra-cellular localization of these variants (FIG. 2C). Quantitativevalues were expressed as either percent positive cells (expressionlevels) within each population or mean fluorescence intensities of thepositive fraction, which reflected content of the corresponding variantprotein in each population (see below).

In blood cells from post-partum mothers, this analysis expectedly showedvery low to undetectable levels of AChE-S and -R on the surface and inthe cytoplasm of red blood cells (RBCs), compatible with AChE-E beingthe variant that is present and active in these cells. Increasedfluorescence, measured by a shift in histogram patterns compared tobackground, reflected the presence of substantial AChE-S and -R levelsin all of the CD45+ populations. The levels of expression of the AChE-Sand AChE-R variants in post-partum peripheral blood granulocytes andmonocytes was higher (both on the surface and in the cytoplasm) than inlymphocytes, which showed low levels on the surface, and somewhat higherlevels in the cytoplasm (FIG. 3). The surface-cytoplasmic distributionof enzymatically active AChE within blood cells thus presentedlineage-specific differences that were altered both during developmentand following the stress of childbirth.

Example 2 Differential Concentrations of AChE Variants within SpecificBlood Cell Types

An independent quantification of AChE variant levels within specificblood cell types was obtained using the BIAcore technology, based onmeasuring the interaction of proteins in cell homogenates withantibodies covalently linked to a carboxymethyl dextran matrix adherentto the surface of a gold leaf sensor [Johnsson (1991) id ibid.].Increases in the refractive index of this sensor were monitored in realtime as the changes in surface plasmon resonance (SPR) angle (FIG. 4A).Non-specific SPR signals obtained in the absence of antibodies weresubtracted, and protein levels were standardized either to the amount oftotal protein or to the number of lysed cells in each preparation,irrespective of their cellular localization (FIG. 4B).

Because cord blood lysates were examined at only one antibodyconcentration and one lysate concentration, the BIAcore measurementscould only reflect relative antigen interaction with the antibodies, butnot absolute affinity values. These relative amounts of each variantwere compared within specific cell types and between the fourhematopoietic cell groups. When applied to newborn cord blood cellextracts, larger signals were detected for AChE-S than for -R.Decreasing concentrations (reflected in resonance units, RU/mg protein)of AChE-S occurred in the order ofgranulocytes>lymphocytes>monocytes>red blood cells. A differentdecreasing order, lymphocytes>monocytes>granulocytes>red blood cells,was calculated per cell, suggesting that the high granulocyteconcentration of AChE-S reflected the high total protein content. AChE-Rsignals, which were generally lower, presented similar decreasing ordersin both measures (lymphocytes>monocytes>granulocytes>red blood cells,FIG. 4B). The BIAcore and flow cytometry methods revealed distinct R:Sratios, e.g. for granulocytes from cord blood (FIGS. 3, 4), perhaps dueto different properties of the two antibodies.

A third evaluation approach involved immunoblot analysis of thecorresponding cell homogenates using the AChE-S specific ASP antibodydisplayed on phage surface (FIG. 4C). This analysis demonstrated severalimmunopositive protein bands in granulocytes and lymphocytes, most ofwhich of smaller size then the predicted full protein. A single rapidlymigrating band appeared in red blood cells, with fainter, similarlymigrating band in myeloid cells. These bands likely reflect proteolyticbreakdown products (or fragments) of the ACHE-S protein in blood cells.Additionally, or alternatively, the cleaved C-terminus of AChE-S[Grisaru (1991) id ibid.] or an immunocompatible variant could beexposed in cell homogenates but not in intact cells.

Example 3 Distinct Splice Variations in Development and Stress

Calculating the AChE-S/AChE-R distributions as percent of positive cellsin each lineage revealed distinct splice variations in development andstress. Most cell populations included significant fractions of cellspositive for both cytoplasmic and surface AChE-S and -R variants, withsignificantly higher numbers of AChE-R expressing cells in all adultmyeloid cell populations than in fetal cells (FIG. 5). The number ofgranulocytes expressing cytoplasmic AChE-R was significantly higher inpost-partum blood (p<0.05), reminiscent of the increase in AChE-R seenin brain neurons under stress [Kaufer (1998) id ibid.; Meshorer et al.(2002) id ibid.; Soreq and Seidman (2001) id ibid.]. However, AChE-S,which is known to adhere to the membranes of brain neurons was expressedon the surface and in the cytoplasm of significantly more blood cells inpost-partum mothers than in non-pregnant women (Students t-test,p<0.05). In addition, post-partum lymphocytes displayed a paradoxicaldecrease in surface AChE-R. The localization of AChE-R to the cellsurface, which was rather surprising in view of its hydrophilicC-terminal peptide, may reflect interaction with as yet unknown proteinvariant(s) [Birikh (2003) id ibid.].

Example 4 Surface and Cytoplasmic AChE-S and AChE-R Contents inPeripheral Blood Cells

High fluorescence intensity from comparative flow cytometry confirmedthe increased protein content of the AChE-R variant compared to AChE-Sof all cell types and sources tested. AChE-R protein content ofgranulocytes, which comprise 70 to 80% of the white blood cellcompartment, were significantly higher in post-partum mothers comparedto cord blood cells (Table 3), reflecting a strikingly differentsplicing pattern for AChE pre-mRNA in fetal blood cells than in adultcells under the post-partum stimulus. Cytoplasmic increases in AChE-Rcontent, observed as larger mean fluorescence intensity values, occurredunder stress in all cell lineages. Stress-induced increases in cellsurface AChE-R appeared in granulocytes and monocytes, but not in redblood cells. In lymphocytes, cell surface AChE-R increased 8-fold fromnewborns to adults but declined under stress (Table 3).

TABLE 3 AChE variant contents in blood cells Mean Fluorescence Intensity(MFI)^(a) % of blood AChE-S AChE-R leukocytes^(b) Cytoplasmic SurfaceCytoplasmic Surface RBCs CB NA 4.7 ± 1.6 1.4 ± 0.1  6.9 ± 0.3  4.1 ± 0.4APB NA 4.8 ± 0.6 1.4 ± 0.3  5.0 ± 0.3  4.7 ± 2.1 PPB NA 4.5 ± 0.4 4.7 ±0.1 18.5 ± 1.2  5.8 ± 0.6 granulocytes CB 39-63 30.1 ± 15.1 19.4 ± 3.3 57.9 ± 2.7 42.8 ± 3.4 APB 43-65 11.1 ± 0.5  6.1 ± 0.3 32.9 ± 3.9 62.9 ±3.5 PPB 69-82 9.1 ± 0.4 30.1 ± 0.4  96 ± 3 138 ± 10 monocytes CB  6-1221.3 ± 0.1  22.3 ± 3.9  42.7 ± 3.0 29.1 ± 1.7 APB  6-12 10.3 ± 0.3  9.5± 0.7 17.8 ± 1.7 27.1 ± 6.4 PPB  6-12 14.7 ± 0.8  21.3 ± 0.7  57.5 ± 2.6  72 ± 5.8 lymphocytes CB 42-62 10.4 ± 1.3  9.7 ± 1.7 16.1 ± 0.5 24 ± 2APB 21-46 4.8 ± 0.3 2.2 ± 0.1  8.2 ± 0.7 183 ± 11 PPB 13-23 5.7 ± 0.310.4 ± 0.2  25.7 ± 1.1 37 ± 2 ^(a)Average of 15 measurements for eachpopulation expressed as mean ± standard error are presented.^(b)Proportions of white blood cells expressed by peripheral bloodleukocyte populations. Significantly different MFI values as comparedwith the other two sources according to t-test (p < 0.05) analysis arepresented in bold face type.

Example 5 Development and Stress-Induced Changes in Lymphocytic AChEVariants

Previous reports attributed lymphocytes' AChE activity to T cells anddescribed its increases with mitogenic stimulation [Szelenyi et al.(1987) Immunol. Lett. 16: 49-54]. Activity was also observed in thethymus [Topilko and Caillou (1985) Blood 66: 891-5], but B lymphocytesdisplayed very low levels of AChE, which decreased with maturation[Szelenyi et al. (1982) Br. J. Haematol. 50: 241-5]. In the presentstudy, CD3+ T cells presented low expression of surface AChE-R in allsamples while CD19+ B cells expression was significantly higher (FIG.6A), suggesting that AChE-R may be relevant for antibody production. Dueto the majority of T cells (about 9:1 to B cells, FIG. 6A, inset), theirsmall signals contributed significantly to the total lymphocyte output.Nevertheless, B cells displayed a significant increase in fluorescenceintensity from newborn cord blood to adults and post-delivery bloodcells, with no change between the latter two fractions (FIG. 6B). Largerlymphocyte fractions expressed surface AChE-R in separate T and B cellpopulations, likely due to the high background staining in the Tlymphocyte fraction (FIG. 6A).

In conclusion, considerably more myeloid cells of the post-partummothers expressed AChE-S and AChE-R than in either control women ornewborns. In contrast, B lymphocytes lost their surface AChE-R withdevelopment and under stress.

Example 6 Fetal AChE-R mRNA Expression Coincides with Myelopoiesis

The in vivo expression of alternative AChE mRNA transcripts (FIG. 7A)was studied by in situ hybridization using paraffin-embedded human fetalsections from different gestational ages (FIG. 7A). AChE mRNAs wereobserved in the aorta-gonad-mesonephric region (AGM), liver, spleen andbone marrow, consistent with the spatiotemporal shifts of hematopoieticembryogenesis and the migration of fetal hematopoiesis through thevarious blood forming tissues (FIG. 7C). Clear changes occurred indeveloping liver, with distinct labeling intensities for each of theAChE mRNA transcripts at different gestational ages (FIG. 7D). At 9weeks gestation, when the liver and spleen are initiating definitivehematopoiesis, the erythrocytic AChE-E mRNA transcript was prominentlydisplayed in the AGM, liver and spleen. Significant levels of thesynaptic transcript (AChE-S mRNA) were found at this time in the AGMregion and liver, but not in spleen, while the AChE-R mRNA variant wasbarely detectable in all hematopoietic tissues. At 16 weeks, duringaccelerated myelopoiesis, AChE-S was elevated in both liver and spleenin agreement with findings of others [Chan (1998) id ibid.]. A decreasein AChE-E mRNA concurrent with an increase in AChE-R mRNA was observedin the liver, suggesting a splicing shift (FIG. 7E). Subsequentdecreases in all AChE mRNA variants were observed until 25 weeks. Thesechanges were concomitant with the switch from primitive hematopoiesis,which is exclusively erythrocytic, to definitive hematopoiesis of alllineages. These results suggest that AChE-R overproduction is causallyassociated with myelopoiesis in vivo.

Example 7 Intra-Partum Cortisol Escalation Associates with IncreasedGranulocytic AChE-R Expression

Cortisol levels were predictably elevated intra-partum as compared to anage matched population of 48 control Caucasian women (36.6±4.2 vs.21.3±11.2 μg/dL, p<0.001; FIG. 8A). Intra-partum serum cortisol levelsshowed direct correlation with WBC counts (Pearson correlation; R=0.55,p=0.04; FIG. 8E). This was accompanied by elevated expression of AChE-Rin the cytoplasm of mature WBC as detected by flow cytometry (p=0.009;FIG. 8F). Direct, significant correlation of cortisol levels with thefraction of AChE-R positive granulocytes (R=0.72, p=0.003; FIG. 8G), butnot monocytes or lymphocytes, was consistent with the predicted role ofAChE-R in post-partum granulocytosis.

Example 8 Sustained Peri-Partum Granulocytosis

To explore the relevance of cholinergic changes for intra-partumgranulocytosis, the peri-partum hematopoietic changes in blood sampleswas studied. Sixteen patients with premature rupture of membranes atterm (PROM, rupture of membranes without uterine contractions) werefollowed, from admission through delivery and post-partum periods(27.08±14.22 and 61.82±15.99 hours post admission, respectively). WBCcounts in these patients were higher than the pre-delivery average andincreased significantly intra-partum (P<0.0001; FIG. 9A). Hemoglobinlevels maintained normal to low range before delivery and decreasedsignificantly intra- and post-partum (P=0.01), compared to the baselinevalues, reflecting blood loss during labor. Platelet counts remainedstable and in the normal range during the entire study period (FIG. 9A).Although WBC counts decreased post-partum, they remained significantlyabove normal ranges (P=0.01)), reflecting increased granulocyte counts(intra-partum: P<0.0001; post-partum: P=0.02). Monocyte and lymphocytecounts remained in the low normal range (FIG. 9B). In vivo parturitionwas therefore considered appropriate for assessing the effects of atransient stressful event on granulocytosis.

Example 9 Granulocytic AChE-R Expression Maintains High Post-PartumLevels

Cortisol levels were high pre-partum (30.6±8.2 vs. 21.3±11.2 μg/dL inage-matched control population, P<0.001), increased intra-partum(32.1±12.2 μg/dL; P<0.001 compared to matched controls), and decreasedsignificantly post-partum (27.2±10.6 μg/dL, P=0.05 compared to theintra-partum values; FIG. 8A) to levels that are not statisticallydifferent than those of the matched control population. Serum AChEactivity increased as compared to controls (21.6±7.2 vs. 5.5±1.9nmole/min/mg protein; p<0.001) and remained significantly elevatedduring the entire period (FIG. 8B). A significant increase was observedin the number of granulocytes expressing cytoplasmic AChE-R, both intra-and post-partum as compared to pre-partum (from 1.7±0.6×10³ cells/μL to5.2±0.5 and 4.9±0.4×10³ cells/μL, respectively, P=0.05; FIG. 9D). Thispattern of expression was not reflected in monocytes or lymphocytes(FIG. 9D), consistent with the idea that AChE-R may have a selectiverole in the prolongation of peri-partum granulocytosis. High serumlevels of AChE-R were found throughout the peri-partum period (FIG. 9D),supporting the notion that serum AChE activity reflected sustainableAChE-R levels, facilitating parturition anxiety [Sklan, E. H. et al.(2004) Proc. Natl. Acad. Sci. USA 101(15): 5512-5517].

Example 10 Parturition Effects on Myeloid Markers

To determine the effect of parturition on the myeloid lineage, appliedflow cytometry to study the expression of CD15 (a marker ofgranulocytes), CD 33 (a marker of early myeloid cells) and CD14 (whichis expressed on myeloid cells and is often used as a marker ofmonocytes) on peripheral blood WBC. CD15 expression on granulocytesdecreased significantly in intra-partum samples (535±287 vs. 294±129MFI, P=0.03; FIG. 9C-9D) and its post-partum levels returned tobaseline, while CD33 expression did not change significantly over theentire period. This may represent a cumulative effect of rapidproduction and release of early myeloid cells from the bone marrow onthe one hand, accompanied by their rapid maturation on the other hand.Additionally, CD14 expression on monocytes did not vary, whilepost-partum CD33 expression decreased significantly (145±89 vs. 91±40MFI, P=0.05; FIG. 9C-9D).

Example 11 Leukocyte AChE-R Contents Positively Associate with PlasmaAChE Activity

Total AChE-R contents in blood cells were evaluated by multiplying themean fluorescence intensity (MFI) per the percent of positive cellsexpressing AChE-R in each cell type (granulocytes, monocytes andlymphocytes) detected by flow cytometry. In each type of circulatingWBC, AChE-R contents correlated with AChE activity in the post-partumplasma (for granulocytes R=0.984, p<0.0001; for monocytes R=0.962,p<0.0001; and for lymphocytes R=0.917, p<0.0001; FIG. 10). Plasma AChEactivity levels thus reflected AChE-R production in each type ofleukocyte.

Example 12 ARP₂₆ Enhances Endogenous ACHE Gene Expression

Assuming a turnover number of 1×10⁴ molecules of AChhydrolyzed/second/AChE subunit, and based on the inventors' previousfindings [Cohen (2003) id ibid.], up to one-half of the AChE-R isC-terminally cleaved in vivo to yield ARP, the AChE-R C-terminalpeptide. Therefore, the measured rates of ACh hydrolysis in the serum ofpost-partum mothers predicted a peptide concentration in the range of5-30 nM. It was further hypothesized that comparable peptideconcentrations are found in the bone marrow, and the potential ex vivoeffects of ARP₂₆ at 0.2, 2.0 and 20 nM on CD34+ progenitors. In situhybridization followed by confocal quantification of the three AChE mRNAvariants revealed increased levels of all AChE mRNA transcripts 24 hoursfollowing the addition of ARP₂₆ to the medium. This was accompanied byincreased cytochemically stainable cellular ACh hydrolytic activityreflecting accumulated AChE protein in the ARP₂₆-treated cultured cells(FIG. 11C). The enhanced activity under physiologically relevantconcentrations of ARP₂₆ reflected an increase in endogenous AChE, sincethe synthetic peptide has no enzymatic capacity. It also provided apossible explanation for the sustained AChE activity in peri-partumsera, since it occurred with 2 nM ARP₂₆ and to a similar extent inmammals exposed to stress-associated cortisol levels [Grisaru (2001) idibid.].

Example 13 ARP₂₆ Potentiates Myelopoiesis in Liquid Cultures

To test the long-term effect of ARP₂₆ on myelopoietic expansion, flowcytometry was used to monitor the development of phenotypically distinctcell populations from human CD34+ hematopoietic stem cells incubatedwith ARP₂₆ over a 2-week period. Peptide controls (ASP₄₀ and PBAN) wereused to explore the specificity of this response. FIGS. 11D-11E andTable 4 present the resultant cell growth and changes in the populationsthat emerged from a typical CD34+ culture. Incubation with ARP₂₆, butnot with cortisol, ASP₄₀ or PBAN, increased the total number of cells. Alarger fraction of committed progenitors (CD34+CD38+) emerged in thepresence of cortisol at stress levels (1.2 μM) as compared to aphysiologically relevant concentration of ARP₂₆ (FIG. 11D-11E); however,the expansion index (the number of viable cells/ml culture divided bythe number of seeded cells) was considerably higher following incubationwith ARP₂₆ (Table 4a). Increases were observed along the entiremyelopoietic differentiation pathway (CD34+CD33+, CD33+CD15−, CD33+CD15+and CD33−CD15+, Table 4b), supporting the notion that ARP₂₆ tiltshematopoiesis towards the myeloid lineage in a cortisol-independentprocess, and particularly expanding the population of mature CD33−CD15+granulocytes (see column CD33−CD15+, Table 4b), inducing increasedgrowth of early GEMM progenitors and producing specifically largenumbers of mature granulocytes. These findings demonstrate that theARP₂₆-induced myelopoiesis in fact leads to an enrichment of thegranulocytic population.

TABLE 4a ARP₂₆ promotes ex vivo cell expansion of cultured CD34+ cellsCell type Cell expansion Treatment index^(a) Control 0.59 ± 0.76Cortisol, 1.2 μM 1.55 ± 0.07 ARP₂₆, 2 nM  5.29 ± 2.52* ASP₄₀, 2 nM  1.9± 0.61 PBAN, 2 nM 1.85 ± 0.96 ^(a)The number of viable cells/ml cultureat day 14 divided by the number of seeded cells (50,000). *P < 0.001

TABLE 4b The effect of various conditions on cultured CD34+ cells. - Thenumbers represent actual cell counts × 10³ of each cell type detectedCell type CD34+ CD34+ CD33+ CD33− Cell CD38+ CD33+ CD15− CD33+ CD15+expansion Committed Committed Immature CD15+ Mature Treatment index^(a)progenitors myeloids myeloids Granulocytes granulocytes Control 1.2 0.50.5 1.4 2.1 4.3 Cortisol 1.6 11.0 10.6 13.0 28.5 15.4 (1.2 nM) ARP₂₆ (2nM) 11.4 29.1 31.9 94.6 64.4 220.6 ASP (2 nM) 2.0 2.5 0.4 0.5 1.9 4.2PBAN (2 nM) 2.1 0 0.1 0.2 0.9 1.8 ^(a)Expansion index is a ratio of thenumber of viable cell/ml culture at day 14 divided by the number ofcells seeded (50,000).

Example 14 AChE-R Supports Pro-Inflammatory Cytokine Release fromMononuclear Cells

Next, the putative mechanism(s) enabling the long term effects of ARP₂₆was addressed. The elevated AChE-R contents and AChE activity in thepost-partum blood predicted peripherally reduced ACh levels, and thesuppressed cholinergic control over the production of pro-inflammatorycytokines by macrophages. The levels of severalinflammation/stress-associated cytokines in the plasma of intra-partummothers were compared to those of non-pregnant women. Elevation wasobserved for IL-1β, IL-6 and TNFα, all known to have pro-inflammatoryand hematopoietic roles, in the post-partum mothers [Hanada andYoshimura (2002) Cytokine Growth Factor Rev. 13: 413-421; Wilson et al.(2002) J. Am. Geriatr. Soc. 50: 2041-2056] (FIG. 12A). To examinewhether this increase could be causally related with AChE-Roverexpression in peripheral white blood cells, 2.5×10⁶ mononuclearcells per mL from adult women controls were incubated with 2 nM ARP(FIG. 12B). Significant increases were observed in the secretion fromthese cell cultures of IL-1β, IL-6 and TNFα 24 hours later, but therewas no change in the release of the anti-inflammatory cytokine IL-8 fromcells incubated with ARP₂₆ as compared with control cells (FIG. 12A anddata not shown). Thus, the post-partum AChE-R overexpression inperipheral nucleated blood cells could be causally associated withselective elevation of pro-inflammatory cytokines.

Example 15 AChE-R Excess is Associated with Impaired Response to LPS

In order to understand the mechanisms of the hematopoietic effect ofprolonged exposure to AChE-R, the transgenic AChE-R (TgR) mouse modelwas used. Basal levels of white blood cell counts (WBC) were similar inboth TgR and FVB/N mice (FIG. 14A-14B). Manual differential of WBCsub-populations showed similar distributions into granulocytes,monocytes and lymphocytes in TgR and FVB/N mice (FIG. 14A-14B). Thesefindings may reflect an equilibrium state of the hematopoietic systemreached by the TgR mice. Therefore it was demanding to expose these miceto an acute stressful event.

LPS was injected intra-peritoneally (IP) in order to induce acuteinflammation WBC counts dropped in both FVB/N and TgR mice. However,counts recovered much faster in TgR mice to reach significantly higherlevels than those of FVB/N mice by 72 hr post LPS injection (p<0.02,n=10, FIG. 14B). Peripheral blood immunophenotyping revealed that whileFVB/N mice had a significant decrease in GR1⁺ (granulocyte) cells, inresponse to LPS injection, the number of GR1⁺ remained unchanged in TgRmice and was significantly higher than FVB/N by 72 h post LPS injection.Both FVB/N and TgR mice had decreased CD11b+ (monocytic) cell counts 24h post LPS injection, although the decrease was steeper in TgR ascompared to FVB/N mice. CD11b+ cell counts recovered almost completelyby 72 h post LPS injection in both FVB/N and TgR mice, TgR miceattaining higher Cd11b+counts, although not reaching a statisticallysignificant value. These data suggest that the early recovery in WBCcounts in TgR in response to inflammatory stress, results from boththeir ability to maintain stable granulocyte counts, in spite of the LPSsuppressive effects, as well as to a rapid renovation of the monocytepool.

Example 16 PU.1 Transcription Factor is Involved in the InflammatoryResponse

To further understand TgR peripheral cell response to inflammatorystress, the dexpression pattern of transcription factors pivotal forhematopoiesis in bone marrow extracts from FVB/N and TgR mice wasevaluated, through real time RT-PCR (FIG. 13).

While the response pattern of LMO2, GATA1, RUNX1 and STAT5 to LPS wassimilar in both FVB/N and TgR mice, PU.1 levels decreased significantlyin FVB/N, but not in TgR mice bone marrow, in response to LPS. At 72 hpost LPS injection, PU.1 levels recovered and even reached higher thanbase-line values in FVB/N mice, but showed only some decrease in TgRmice.

Example 17 AChE-R is Expressed in Bone Marrow and Blood of TgR Mice

The inventors' previous reports suggest that a stress-induced switchfrom production of AChE-S to the -R variant elevates soluble AChE-Rlevels [Pick (2004) id ibid.]. Thus, it was hypothesized that this shiftmay reduce circulating ACh and the nicotinic α7 ACh control overpro-inflammatory cytokine production [Tracey K. J. (2002) id ibid.],driving hematopoietic progenitor cell expansion, as previously described[Grisaru (2001) id ibid.] (FIG. 16A). TgR mice were then used as a modelof chronic splicing shift towards the AChE-R, over the AChE-Stranscript. Using DNA primers specific for human AChE intron 4, humanAChE-R mRNA was detected in the BM of TgR mice but not in strain-matchedFVB/N mice or in the TgS mice over-expressing the AChE-S variant, (FIG.16B), reconfirming the continued activity of the transgene inhematopoietic cells.

Example 18 AChE-R Excess is Associated with Elevated Basal andPost-Stress Platelet Counts

As mentioned before, basal levels of WBC were similar in TgR and FVB/Nmice (FIGS. 14A-14B and 17B). Basal levels of thrombopoietin (TPO) weresimilar in both TgR and FVB/N mice (FIG. 17A), whereas platelet countswere significantly higher in TgR mice (894±87 Vs 1051±160×10⁹/mL,p<0.001, n=25, FIG. 17C). Since manual differential of WBCsub-populations showed similar distributions into granulocytes,monocytes and lymphocytes in TgR and FVB/N mice (FIGS. 14A-14B and 17B),the results with the platelets reflect selective thrombocytosis underchronic AChE-R overexpression.

After ip LPS injection RBC counts predictably dropped up to 72 hrspost-LPS (FIG. 16) (in control FVB/N but not TgR mice. WBC dropped inboth strains, but counts recovered considerably faster in TgR micereaching significantly higher levels than those of FVB/N control mice by72 hr post LPS injection (p<0.02, n=10, FIGS. 16B and 16D). Plateletcounts in FVB/N control mice dropped significantly, as expected, tothrombocytopenic levels between 24 and 72 hrs, while in TgR mice theplatelet counts were only slightly reduced and returned to normal valueswithin 72 hrs (p<0.001, n=10, FIG. 16C).

Example 19 AChE-R Over-Expression Modulates TPO and InflammatoryCytokine Levels

To further study the observation of elevated platelet counts in TgRmice, TPO concentrations were measured in the plasma and BM cellextracts from TgR and FVB/N mice. TPO concentrations were significantlyhigher in both BM and plasma of TgR mice (p=0.013, 0.04 respectively,compared to FVB/N control mice (FIGS. 17A and 17B), consistent with thenotion that these mice can serve as a model of chronic inflammation[Stohlawetz (1999) id ibid.; Kaser A. et al. (2001) Blood 98: 2720-2725;Zahorec R. (2001) Bratisl. Lek. Listy. 102:5-14]. TgR mice BM had higherTPO levels 24 hrs post LPS injection (p=0.002) followed by lower TPOlevels at 72 hrs (p=0.02, n=10, FIG. 3A), as compared with FVB/N mice.In plasma, the high basal TPO levels were maintained 24 hrs post LPSinjection (p=0.01, n=10). However, at this time point the FVB/N miceplasma TPO levels were significantly higher than TgR mice possibly dueto corresponding dramatic drop in platelet numbers (FIG. 17B). TPOlevels decreased slightly but remained elevated in both mouse strains at72 hrs (FIG. 17B).

Example 20 AChE-R Over-Expression is Associated with ModifiedInflammatory Cytokine Levels

To study the possible effects of AChE-R in the inflammatory reaction,the inventors measured the levels of inflammatory cytokines in plasmaand BM extracts of TgR and FVB/N mice. IL-6, but not TNFα levels werefound to be significantly elevated in the plasma of TgR mice as wasAThCh hydrolyzing activity compared with FVB/N control mice, suggestingthat AChE catalytic activity might be involved in modified inflammatorycontrol (FIG. 15C).

The inflammatory response of TgR mice was further evaluated by measuringthe levels of TNFα and IL-6, in bone marrow cell extracts and plasma,and at different time points post injection of LPS. TgR mice showedsignificantly higher plasma levels of TNFα 2 hrs post LPS injection(34±231 pg/mL, p<0.04, n=10) but significantly lower levels in BM, ascompared to FVB/N mice (120±66 Vs 334±81, p<0.01, n=10) (Table 5A)possibly because the main production TNFα is in peripheral blood. IL-6levels were comparable in both TgR and FVB/N mice after LPS injection(Table 5A), indicating again a pre-existing active inflammatory state inthe TgR strain.

TABLE 5A Inflammatory cytokine levels post LPS TNFα (pg/ml) ^(a) IL-6(pg/ml) ^(a) plasma BM Plasma BM TgR 834 ± 231 120 ± 66 1418 ± 62 507 ±135 FVB/N 538 ± 217 334 ± 81 1378 ± 40 498 ± 331 P 0.04 0.01 NS NS ^(a)TNFα and IL-6 levels were measured 2 hours post-LPS injection.

In addition to their high baseline AChE catalytic activity (FIG. 15C),TgR mice responded to LPS injections by a further significant increasein bone marrow AChE catalytic activity 24 hrs post LPS injection(p=0.0004, n=10), but not at other time points (Table 5B).

TABLE 5B AThCh hydrolysing activity/min/mg of protein post LPS BM PlasmaLPS (hrs post) LPS (hrs post) 24 72 24 72 TgR 14.2 ± 4.3 13.1 ± 1.8 31.7± 12.8 7.7 ± 0.4 FVB/N  6.3 ± 1.9 11.0 ± 0.7 27.0 ± 13.1 9.3 ± 1.0 P0.00004 NS NS NS Note: AChE catalytic activity assessed by its AThChhydrolyzing activity/mim/mg protein was measured in plasma and bonemarrow extracts of LPS-injected mice (n = 10) Shown are averageconcentrations ± SD in plasma or BM proteins.

Example 21 Enhanced Proliferative Potential in Bone Marrow Progenitorsfrom TgR Mice

The proliferating potential of BM progenitor cells was evaluated byclonogenic assays using growth factors to support the development of thespecific hematopoietic lineages. Colonies were classified as colonyforming units—megakaryocyte (CFU-Mk), CFU-granulocyte/macrophage(CFU-GM) or CFU-granulocyte/erythrocyte/monocyte/megakaryocyte(CFU-GEMM) and were counted 10 to 14 days after plating. TgR mice showedsignificantly higher baseline numbers of CFU-Mk, -GM and -GEMMhematopoietic progenitor cells as compared to FVB/N controls (p≦0.003,n=12, FIGS. 18A-21C). Following LPS injection, TgR mice maintainedsignificantly higher number of megakaryocyte progenitors (p<0.0002,n=12, FIG. 18A). In FVB/N mice, the number of CFU-GM, was significantlyelevated at 24 hr post-LPS, a response previously described [Peterson(1992) id ibid.; Yokochi (1985) id ibid.] (p=0.01, n=12, FIG. 18B) butdecreased noticeably by 48 hr, while TgR CFU-GM numbers decreased 48 hrpost LPS injection but remained significantly higher than FVB/N (p=0.03,n=12, FIG. 18B). The increase in CFU-GM in TgR mice was less dramaticthan in FVB/N control mice perhaps caused by fatigue of myeloidprogenitor cells due to chronic exposure to ACHE-R. TgR and FVB/N miceshowed similar post-LPS numbers of multipotential CFU-GEMM (NS, n=12,FIG. 18C).

Example 22 AChE-R Over-Expression Associates with ElevatedMegakaryocytic PKCε

AChE-R was reported to interact with the scaffold protein RACK1 and withits target, protein kinase C βII (PKC βII) [Birikh K. R. et al. (2003)Proc. Natl. Acad. Sci. USA 100:283-288; WO 00/73427] or PKC ε [Perry C.et al. (2004) Neoplasia 6(3):279-86]. PKCε has been implicated in theprogramming of megakaryocytic lineage commitment and potentiates thetranscription factor GATA-1 [Racke F. K. et al. (2001) J. Biol. Chem.276:522-528]. To study a potential AChE-R/PKCε/RACK1 interaction inmegakaryocytes, AChE-R, PKCε and RACK1 were detected in BM smears of TgRand FVB/N mice (FIG. 19A-19F).

Megakaryocytes were detected in BM smears by the May-Grünwald staining(FIG. 19A). TgR megakaryocytes predictably expressed higher AChE-Rlabeling then megakaryocytes from FVB/N mice (212.3±15.0 Vs 130.9±18.3luminescence units, p<10⁻¹¹, n=50, FIGS. 19B, 19F and Table 3).Intriguingly, RACK1 labeling intensity was discernable, althoughinsignificantly elevated in TgR megakaryocytes, as compared to FVB/Nmice (162.3±49.2 Vs 153.4±21.0, NS, n=50, FIGS. 19C, 19F and Table 6).No differences in the number of PKC ε-labeled megakaryocytes weredetected in TgR mice (data no shown), nevertheless, the intensity of PKCε labeling was significantly higher as compared to FVB/N mice(187.7±22.2 Vs 160.9±19.7 luminescence units, p<10⁻⁵, n=50, FIGS. 19D,19F and Table 6). Thus, AChE-R interaction with RACK1 and with PKCεemerged as a putative mechanism for increased intracellular signaling inTgR megakaryocytes.

TABLE 6 Luminescence intensity of human AChE-R, RACK1 and PKCε inmegakaryocytes. Antibody FVBN TgR p values No Antibody 116.8 ± 8.7 122.4 ± 11.8 NS Hu AChE-R 130.94 ± 18.26 212.3 ± 15.0 1 × 10⁻¹¹ RACK1153.4 ± 21.0 162.3 ± 49.2 NS PKCε 160.9 ± 19.7 187.8 ± 22.7 3 × 10⁻⁶ 

Note: Luminescence levels (from 1, low luminescence to 220, bright) weredetermined using a upright Zeiss microscope, ImagePro™ image capture andAdobe Photoshop V 5.5 analysis for each megakaryocyte stained in thebone marrow smears (n=50 per antibody). NS=Not significant. Backgroundstaining was detected by incubation with no primary antibody.

Example 23 AChE-R Potentiates Engraftment Potential in NOD/SCID Mice

TgR mice elevated platelet counts and increased megakaryocyte growthpotential was suggestive to determine whether AChE-R, or its cleavablepeptide ARP, can improve engraftment of transplanted BM cells andrecovery from thrombocytopenia in a NOD/SCID mouse transplantationmodel. Human CB CD34⁺ cells were primed for 2 hrs prior to injectionwith ARP₂₆, a synthetic peptide comprised of 26 amino acids of theC-terminal sequence of AChE-R or ASP₄₀ a 43 amino acid sequence of the Cterminus of AChE-S.

The ARP concentration chosen (2 nM) was previously determined to bemaximal for stimulating hematopoietic stem cell proliferation [Deutsch(2002) id ibid.] Human CB CD34+ cells (1×10⁵) were injected into mice 24hours post irradiation. Cells were either primed and supplemented withARP₂₆ or primed and supplemented with ASP₄₀ or untreated (control). Micewere sacrificed 6 weeks post-transplantation and single cell suspensionsfrom BM extracted from the femur bones assessed for the presence ofhuman hematopoietic cells Monoclonal antibodies against human CD45, CD34and CD41 were used to assess engraftment efficacy of the transplantedhuman cells. Fractions of human CD34⁺ cells in the BM of NOD/SCID micepost transplant were similar in all groups (FIG. 20A). However,significantly more human CD45⁺ cells were found in the BM of NOD/SCIDmice injected with ARP₂₆ together with ARP₂₆-primed CD34⁺ cells (p=0.02,n=12, 16 and 8 mice, respectively, FIG. 20A). Fractions of humanmegakaryocytes (CD41⁺) were higher in the BM of NOD/SCID mice thatreceived ARP₂₆-primed cells as compared with ASP₄₀-primed or non-primedhuman cells (p=0.03, n=12, 16 and 8 mice, respectively, FIG. 20A). Theseresults demonstrate a significantly better engraftment of transplantedprimed human CD34⁺ cells when injected with ARP₂₆ as compared withnon-ARP₂₆ treated cells.

Quantitative PCR with human specific probes was used to assess therelative presence of human DNA in the BM of NOD/SCID mice. Significantdifferences could be observed between mice transplanted withARP₂₆-primed CD34⁺ cells as compared to cells primed with ASP₄₀ ornon-primed cells (p=0.015, FIG. 20B).

Example 24 Transplantation of Cells Expanded Ex Vivo with ARP₂₆Increased Human Platelet Production in NOD/SCID Mice

In an attempt to improve platelet recovery in NOD/SCID mice the numberof committed megakaryocyte progenitor cells in the stem cell graft wereexpanded ex-vivo. CD34⁺ cells were incubated for 10 days in mediumsupplemented with 10% plasma and one of the following combinations:ARP₂₆, (2 nM) ASP₄₃ (2 nM), TPO (10 ng/ml) and SCF (50 ng) (growthfactors optimal for megakaryocyte commitment) or no growth factorsupplement (control). CD34+ cells are known to differentiate in cultureproducing many committed progenitors, but have reduced capacity forlong-term engraftment in NOD/SCID mice. Therefore, freshly isolatedCD34⁺ cells are needed to enable long-term engraftment [Guenechea G. etal. (1999) Blood 93:1097-1105; Li K. et al. (1999) Br. J. Haematol.104:178-185]. For this reason a mixture of cultured CD34⁺ cells (between1-2×10⁵) was injected together with 100,000 fresh CB CD34⁺ cells. Thecultured CD34⁺ cells, being more mature were expected to facilitate thecapacity for early platelet production. Early engraftment (2-3 weekspost-transplant) and late engraftment (4 and 6 weeks post-transplant)were analyzed. Incubating CD34⁺ cells with ARP₂₆, ASP₄₀ or TPO and SCFdid not augment engraftment of human CD45⁺, CD34⁺ or CD41⁺ cells (FIG.21A) however, it enabled to test whether the injected differentiatedcells affected platelet production. Full blood cell counts wereperformed on the transplanted NOD/SCID mice and the presence of humanplatelets monitored. Although significant differences were not found,probably due to the small sample number of mice (n=6), NOD/SCID micethat received cells expanded with ARP₂₆ yielded higher human plateletnumbers, both early (between 2 and 3 weeks post-transplant) (mean=1.26control Vs 3.29 ARP₂₆ expanded Vs 0.94 ASP₄₀ expanded Vs 1.61×10⁶/mlTPO/SCF expanded group, FIG. 21B) and at the late transplanted stage(mean=5.85 control Vs 17.70 ARP₂₆ expanded Vs 6.39 ASP₄₀ expanded Vs3.44×10⁶/ml TPO/SCF expanded group, FIG. 21B). These observations werecompatible with the hypothesis that the injected differentiatedmegakaryocytes facilitated platelet production in the engrafted mice andthat the enhanced AChE-R production by these cells support a shifttowards megakaryocytopoiesis, which culminated in higher platelet countsat the later test time.

1. A method of inducing production of granulocytes in a subject in needthereof, comprising administering a therapeutically-effective amount ofan acetylcholinesterase (AChE)-derived peptide, or functional fragments,derivatives, or a composition thereof to said subject, wherein saidAChE-derived peptide is denoted by SEQ ID NO:1.
 2. A method of treatmentof conditions that trigger low cell count of granulocytes, comprisingadministering a therapeutically-effective amount of anacetylcholinesterase (AChE)-derived peptide, or functional fragments,derivatives, or a composition thereof to a subject in need, wherein saidAChE-derived peptide is denoted by SEQ ID NO:1.
 3. A method of inducingblood cells to produce cytokines, comprising obtaining said cells from asubject in need of cytokine-producing blood cells, isolating immaturecells, and contacting said cells with an acetylcholinesterase(AChE)-derived peptide, or functional fragments, derivatives, or acomposition thereof, wherein said AChE-derived peptide is denoted by SEQID NO:1.